Human antibodies to fel D1 and methods of use thereof

ABSTRACT

The present invention provides antibodies that bind to the cat allergen, Fel d1, compositions comprising the antibodies, nucleic acids encoding the antibodies and methods of use of the antibodies. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to Fel d1. The antibodies of the invention are useful for binding to the Fel d1 allergen in vivo, thus preventing binding of the Fel d1 allergen to pre-formed IgE on the surface of mast cells or basophils. In doing so, the antibodies act to prevent the release of histamine and other inflammatory mediators from mast cells and/or basophils, thus ameliorating the untoward response to the cat allergen in sensitized individuals. The antibodies of the invention may also be useful for diagnostic purposes to determine if a patient is allergic to the Fel d1 cat allergen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/732,578, filed Jun. 5, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/875,401 filed May 2, 2013, now U.S. Pat. No.9,079,948, which claims the benefit under 35 U.S.C § 119(e) of U.S.Provisional Application Ser. Nos. 61/642,083, filed May 3, 2012;61/718,044, filed Oct. 24, 2012, and 61/783,312, filed Mar. 14, 2013,all of which are herein specifically incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention is related to human antibodies and antigen-bindingfragments of human antibodies that specifically bind to the cat allergenFel d1, therapeutic compositions comprising the antibodies and methodsof using those antibodies.

STATEMENT OF RELATED ART

The Fel d1 protein is a secreted cat protein, which belongs to thesecretoglobin family of small disulfide linked heterodimeric proteinsfound only in mammals (Klug, J. et al. (2000), Ann. N.Y. Acad. Sci.923:348-354). It is the major cause of cat allergies in humans(Platts-Mills, T. A., et al. (1997), J. Allergy Clin. Immunol.100:S2-S24). About 90-95% of patients allergic to cats have an IgEresponse to the Fel d 1 protein (van Ree, et al. (1999), J. AllergyClin. Immunol. 104:1223-1230). The symptoms in a patient who experiencesan allergic response to Fel d1 can range from mild rhinitis andconjunctivitis to life-threatening asthmatic responses. Fel d1 isproduced by sebaceous glands and squamous glands and squamous epithelialcells and is transferred to the pelt by licking and grooming(Bartholome, K. et al. (1985), J. Allergy Clin. Immunol. 76:503-506;Charpin, C. et al. (1991), J. Allergy Clin. Immunol. 88:77-82;Dabrowski, A. J. (1990), et al. J. Allergy Clin. Immunol. 86:462-465).It is also present in the salivary, perianal and lachrymal glands(Andersen, M. C., et al. (1985), J. Allergy Clin. Immunol. 76:563-569;van Milligen, F. J. (1992), et al., Int. Arch. Allergy Appl. Immunol.92(4):375-378) and the principal reservoirs appear to be the skin andthe fur (Mata, P. et al. (1992), Ann. Allergy 69(4):321-322).

Natural Fel d1 is an approximately 18 kDa heterodimeric glycoprotein.Each heterodimer comprises two polypeptide chains, which are covalentlylinked by three inter-chain disulfide bonds and which are encoded by twoseparate genes (Duffort, O A, et al., (1991), Mol. Immunol. 28:301-309;Morgenstern, J P, et al., (1991), PNAS 88:9690-9694; Griffith, I. J., etal. (1992), Gene 113:263-268; Kristensen, A. K. et al. (1997), Biol.Chem. 378:899-908). Chain 1 comprises 70 amino acid residues and chain 2comprises about 90-92 amino acid residues. Structurally the two chainsare similar, but have only 10-15% sequence identity (Kaiser, L. et al.(2003), J. Biol. Chem. 278(39):37730-37735). Although each chain issometimes individually referred to as Fel d1, both chains are needed forthe full protein allergen.

The Fel d1 protein is of an unknown function to the animal but causes anIgG or IgE reaction in sensitive humans (either as an allergic orasthmatic response). Although other cat allergens are known, includingFel d2 (albumin) and Fel d3 (cystatin), 60% to 90% of the anti-cat IgEproduced is directed against Fel d1 (Leitermann, K. et al., (1984), JAllergy Clin. Immunol. 74:147-153; Lowenstein, H. et al., (1985),Allergy 40:430-441; van Ree, R. et al., (1999), J. Allergy Clin.Immunol. 104:1223-1230; Ichikawa, K. et al., (2011), Clin. Exp. Allergy,31:1279-1286).

Immunoglobulin E (IgE) is responsible for type 1 hypersensitivity, whichmanifests itself in allergic rhinitis, allergic conjunctivitis, hayfever, allergic asthma, bee venom allergy, and food allergies. IgEcirculates in the blood and binds to high-affinity FcεR1α receptors forIgE on basophils and mast cells. In most allergic responses, theallergens enter the body through inhalation, ingestion, or through theskin. The allergen then binds to preformed IgE already bound to the highaffinity receptor on the surfaces of mast cells and basophils, resultingin cross-linking of several IgE molecules and triggering the release ofhistamine and other inflammatory mediators causing the various allergicsymptoms.

The treatment for allergies includes steroids for suppressing the immuneactivity and bronchial dilators for relieving asthma symptoms.Desensitization therapy is also used for severely allergic patients.Peptide vaccine combinations have been tested for desensitizingindividuals to particular allergens, e.g. Fel d1 (See US2010/0239599A1and EP2380591A2). Antibodies have been proposed as a treatment forallergies, since they may be able to block the entry of allergenicmolecules into the mucosal tissues, or may bind the allergen before ithas the opportunity to bind to the IgE bound to the high affinityreceptor on mast cells or basophils, thus preventing the release ofhistamine and other inflammatory mediators from these cells.

U.S. Pat. No. 5,670,626 describes the use of monoclonal antibodies forthe treatment of IgE-mediated allergic diseases such as allergicrhinitis, allergic asthma, and allergic conjunctivitis by blocking thebinding of allergens to the mucosal tissue. U.S. Pat. No. 6,849,259describes the use of allergen-specific antibodies to inhibit allergicinflammation in an in vivo mouse model of allergy. Milk-based andegg-based antibody systems have been described. For example,US20030003133A1 discloses using milk as a carrier for allergens forinducing oral tolerance to cat dander and other allergens. Compositionsand methods for reducing an allergic response in an animal to anallergen in the environment through use of a molecule that inhibits theability of the allergen to bind to mast cells was described inUS2010/0143266. Other antibodies to Fel d1 were described by de Grootet. al. (de Groot et. al., (1988), J. Allergy Clin. Immunol.82:778-786).

BRIEF SUMMARY OF THE INVENTION

The invention provides fully human monoclonal antibodies (mAbs) andantigen-binding fragments thereof that bind specifically to the catallergen, Fel d1. Such antibodies may be useful to bind the Fel d1allergen in vivo following exposure of a sensitized patient to the catallergen, and as such, may act to either promote clearance of Fel d1 orto block the binding of the allergen to pre-formed IgE on the surface ofmast cells or basophils. By doing so, the antibodies of the inventionmay prevent the release of histamine or other inflammatory mediatorsfrom mast cells or basophils, thereby preventing or diminishing theuntoward effects observed in patients sensitized to the cat allergen. Incertain embodiments, the antibodies may be capable of reducing,minimizing, or preventing at least one symptom in a patient sensitive tothe Fel d1 cat allergen, such as sneezing, congestion, nasal blockage,coughing, wheezing, bronchoconstriction, rhinitis, or conjunctivitis. Incertain embodiments, the antibodies may be capable of preventing evenmore serious in vivo complications associated with exposure to the catallergen in sensitized individuals, such as asthmatic responses,anaphylaxis, or even death.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., (2000), J. Immunol. 164:1925-1933).

A first aspect of the invention provides an isolated human monoclonalantibody or antigen-binding fragment thereof that binds specifically toFel d1.

In one embodiment, the antibody or antigen binding fragment thereof isan isotype other than an IgA isotype.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof has an isotype selected from the group consisting of anIgG1, an IgG2 and an IgG4.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof binds specifically to Fel d1 with a K_(D) equal to orless than 10⁻⁶ M. In one embodiment, the isolated human antibody orantigen-binding fragment thereof binds specifically to Fel d1 with aK_(D) equal to or less than 1.8 nM.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises the three heavy chain CDRs (HCDR1, HCDR2 andHCDR3) contained within any one of the heavy chain variable region(HCVR) sequences selected from the group consisting of SEQ ID NOs: 2,18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242,258, 274, 290, 306, 322, 338, 354, 370 and 460; and the three lightchain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of thelight chain variable region (LCVR) sequences selected from the groupconsisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154,170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378 and468. Methods and techniques for identifying CDRs within HCVR and LCVRamino acid sequences are well known in the art and can be used toidentify CDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,(1997), J. Mol. Biol. 273:927-948; and Martin et al., (1989), Proc.Natl. Acad. Sci. USA 86:9268-9272. Public databases are also availablefor identifying CDR sequences within an antibody.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises the three heavy chain CDRs (HCDR1, HCDR2 andHCDR3) contained within any one of the heavy chain variable region(HCVR) sequences selected from the group consisting of SEQ ID NOs: 18,66, 130, 162, 242, 306, 322, 370 and 460; and the three light chain CDRs(LCDR1, LCDR2 and LCDR3) contained within any one of the light chainvariable region (LCVR) sequences selected from the group consisting ofSEQ ID NOs: 26, 74, 138, 170, 250, 314, 330, 378 and 468.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises a HCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114,130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338,354, 370 and 460.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises a HCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 18, 66, 130, 162, 242, 306,322, 370 and 460.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises a LCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106,122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330,346, 362, 378 and 468.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises a LCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 26, 74, 138, 170, 250, 314,330, 378 and 468.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises: (a) a HCVR having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82,98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306,322, 338, 354, 370 and 460; and (b) a LCVR having an amino acid sequenceselected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90,106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314,330, 346, 362, 378 and 468.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises: (a) a HCVR having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 18, 66, 130, 162, 242,306, 322, 370 and 460; and (b) a LCVR having an amino acid sequenceselected from the group consisting of SEQ ID NO: 26, 74, 138, 170, 250,314, 330, 378 and 468.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises:

-   -   (a) a HCDR1 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100,        116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308,        324, 340, 356, 372 and 462;    -   (b) a HCDR2 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102,        118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310,        326, 342, 358, 374 and 464;    -   (c) a HCDR3 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104,        120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312,        328, 344, 360, 376 and 466;    -   (d) a LCDR1 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108,        124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316,        332, 348, 364, 380 and 470;    -   (e) a LCDR2 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110,        126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318,        334, 350, 366, 382 and 472; and    -   (f) a LCDR3 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112,        128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320,        336, 352, 368, 384 and 474.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises:

-   -   (a) a HCDR1 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 20, 68, 132, 164, 244, 308,        324, 372 and 462;    -   (b) a HCDR2 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 22, 70, 134, 166, 246, 310,        326, 374 and 464;    -   (c) a HCDR3 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 24, 72, 136, 168, 248, 312,        328, 376 and 466;    -   (d) a LCDR1 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 28, 76, 140, 172, 252, 316,        332, 380 and 470;    -   (e) a LCDR2 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 30, 78, 142, 174, 254, 318,        334, 382 and 472; and    -   (f) a LCDR3 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 32, 80, 144, 176, 256, 320,        336, 384 and 474.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises a HCVR/LCVR amino acid sequence pair selectedfrom the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58,66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186,194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314,322/330, 338/346, 354/362, 370/378 and 460/468.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof comprises a HCVR/LCVR amino acid sequence pair selectedfrom the group consisting of SEQ ID NOs: 18/26, 66/74, 130/138, 162/170,242/250, 306/314, 322/330, 370/378 and 460/468.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 comprises a HCVR/LCVR amino acidsequence pair selected from the group consisting of SEQ ID NOs: 18/26,66/74, 130/138 and 162/170.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 comprises the HCVR/LCVR aminoacid sequence pair selected from the group consisting of SEQ ID NOs:18/26 and 322/330.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 comprises the HCVR/LCVR aminoacid sequence pair selected from the group consisting of SEQ ID NOs:18/26 and 306/314.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 comprises the HCVR/LCVR aminoacid sequence pair selected from the group consisting of SEQ ID NOs:18/26 and 370/378.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 comprises the HCVR/LCVR aminoacid sequence pair selected from the group consisting of SEQ ID NOs:242/250 and 306/314.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 comprises the HCVR/LCVR aminoacid sequence pair selected from the group consisting of SEQ ID NOs:242/250 and 322/330.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds specifically to Fel d1 interacts with atleast one amino acid sequence selected from the group consisting ofamino acid residues ranging from about position 15 to about position 24of SEQ ID NO: 396; amino acid residues ranging from about position 85 toabout position 103 of SEQ ID NO: 396; amino acid residues ranging fromabout position 85 to about position 104 of SEQ ID NO: 396; and aminoacid residues ranging from about position 113 to about position 116 ofSEQ ID NO: 396.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with amino acidresidues ranging from about position 15 to about position 24 of SEQ IDNO: 396.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with amino acidresidues ranging from about position 85 to about position 103 of SEQ IDNO: 396.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with amino acidresidues ranging from about position 85 to about position 104 of SEQ IDNO: 396.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with amino acidresidues ranging from about position 113 to about position 116 of SEQ IDNO: 396.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with at least one aminoacid sequence selected from the group consisting of SEQ ID NO: 402, 403,404 and 412.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with SEQ ID NO: 402.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with SEQ ID NO: 403.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with SEQ ID NO: 404.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with SEQ ID NO: 426.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof which binds to Fel d1 interacts with SEQ ID NO: 412.

In one embodiment, the isolated human antibody or antigen bindingfragment thereof that interacts with SEQ ID NOs: 402, 403, 404 and/or426, comprises the three HCDRs contained in the heavy chain variableregion of SEQ ID NO: 18 and the three LCDRs contained in the light chainvariable region of SEQ ID NO: 26.

In one embodiment, the isolated human antibody or antigen bindingfragment thereof that interacts with SEQ ID NOs: 402, 403, 404 and/or426, comprises a HCDR1 of SEQ ID NO: 20; a HCDR2 of SEQ ID NO: 22; aHCDR3 of SEQ ID NO: 24; a LCDR1 of SEQ ID NO: 28; a LCDR2 of SEQ ID NO:30 and a LCDR3 of SEQ ID NO: 32.

In one embodiment, the isolated human antibody or antigen bindingfragment thereof that interacts with SEQ ID NO: 412 comprises the threeHCDRs contained in the heavy chain variable region of SEQ ID NO: 306 andthe three LCDRs contained in the light chain variable region of SEQ IDNO: 314.

In one embodiment, the isolated human antibody or antigen bindingfragment thereof that interacts with SEQ ID NO: 412 comprises a HCDR1 ofSEQ ID NO: 308; a HCDR2 of SEQ ID NO: 310; a HCDR3 of SEQ ID NO: 312; aLCDR1 of SEQ ID NO: 316; a LCDR2 of SEQ ID NO: 318 and a LCDR3 of SEQ IDNO: 320.

In one embodiment, the human antibody or antigen binding fragmentthereof that binds Fel d1 comprises the HCDR1, HCDR2 and HCDR3 aminoacid sequences of SEQ ID NO: 20, 22 and 24, respectively and LCDR1,LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 28, 30 and 32,respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof that binds to Fel d1 comprises the HCDR1, HCDR2 and HCDR3 aminoacid sequences of SEQ ID NO: 68, 70 and 72, respectively and LCDR1,LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 76, 78 and 80,respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof that binds to Fel d1 comprises the HCDR1, HCDR2 and HCDR3 aminoacid sequences of SEQ ID NO: 132, 134 and 136, respectively and LCDR1,LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 140, 142 and 144,respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof that binds to Fel d1 comprises the HCDR1, HCDR2 and HCDR3 aminoacid sequences of SEQ ID NO: 164, 166 and 168, respectively and LCDR1,LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 172, 174 and 176,respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof that binds to Fel d1 comprises the HCDR1, HCDR2 and HCDR3 aminoacid sequences of SEQ ID NO: 244, 246 and 248, respectively and LCDR1,LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 252, 254 and 256,respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof that binds to Fel d1 comprises the HCDR1, HCDR2 and HCDR3 aminoacid sequences of SEQ ID NO: 308, 310 and 312, respectively and LCDR1,LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 316, 318 and 320,respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof that binds to Fel d1 comprises the HCDR1, HCDR2 and HCDR3 aminoacid sequences of SEQ ID NO: 324, 326 and 328, respectively and LCDR1,LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 332, 334 and 336,respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof that binds to Fel d1 comprises the HCDR1, HCDR2 and HCDR3 aminoacid sequences of SEQ ID NO: 372, 374 and 376 respectively and LCDR1,LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 380, 382 and 384,respectively.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that binds to Fel d1,wherein the antibody or fragment thereof exhibits one or more of thefollowing characteristics: (i) comprises a HCVR having an amino acidsequence selected from the group consisting of SEQ ID NO: 18, 66, 130,162, 242, 306, 322, 370 and 460, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; (ii) comprises a LCVR having an amino acid sequenceselected from the group consisting of SEQ ID NO: 26, 74, 138, 170, 250,314, 330, 378 and 468, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; (iii) comprises a HCDR3 domain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 24, 72, 136, 168, 248,312, 328, 376 and 466, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a LCDR3 domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 32, 80, 144, 176, 256, 320, 336, 384and 474, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; (iv)comprises a HCDR1 domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 20, 68, 132, 164, 244, 308, 324, 372 and462, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a HCDR2domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 22, 70, 134, 166, 246, 310, 326, 374 and 464, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a LCDR1 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 28, 76, 140, 172, 252, 316, 332, 380 and 470, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a LCDR2 domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 30, 78,142, 174, 254, 318, 334, 382 and 472, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; (v) binds to Fel d1 with a K_(D) equal toor less than 10⁻⁶ and preferably equal to or less than 10⁻⁹; (vi)demonstrates efficacy in at least one animal model of anaphylaxis orinflammation; or (vii) competes with a reference antibody for binding toFel d1.

In one embodiment, a “reference antibody” may include, for example,antibodies having a combination of heavy chain and light chain aminoacid sequence pairs selected from the group consisting of 18/26, 66/74,130/138, 162/170, 242/250, 306/314, 322/330, 370/378 and 460/468.

In one embodiment, the fully human monoclonal antibody or antigenbinding fragment thereof that binds to Fel d1 comprises a HCDR1 sequencecomprising the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:386) whereinX¹ is Gly, X² is Phe, Tyr or Gly, X³ is Thr or Ser, X⁴ is Phe or Ile, X⁵is Ser, Arg, Thr, or Asn, X⁶ is Asn, Thr, Asp, or Ser, X⁷ is Tyr, and X⁸is Asn, Tyr, or Ala; a HCDR2 sequence comprising the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO: 387), wherein X¹ is Ile, X² is Tyr,Ser, or Asn, X³ is Tyr, Ser, Gly, Pro, or Asp, X⁴ is Asp, Arg, or Ser,X⁵ is Gly, Val, or Ser, X⁶ is Ser, Gly, Arg, or Tyr, X⁷ is Tyr, Arg,Thr, Ser, or Asn, and X⁸ is Ile, Thr, Ala, Ser, or absent; a HCDR3sequence comprising the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶ (SEQ ID NO: 388),wherein X¹ is Ala, X² is Lys or Arg, X³ is Arg, Gly, His, Ser, Asp, Leu,or Thr, X⁴ is Thr, Pro, Arg, Gly, or Glu, X⁵ is Leu, Val, Gly, Lys, Tyr,or Asn, X⁶ is Ser, Arg, Thr, Ala, Tyr, Phe, or Trp, X⁷ is Tyr, Gly, Arg,Ala, Asn, Asp, His, or Asn, X⁸ is Tyr, Thr, Ser, or His, X⁹ is Val, Ser,Ala, Phe, Pro, or absent, X¹⁰ is Met, Gly, Asp, Pro, Val, or absent, X¹¹is Asp, Tyr, Ser, Gly, Phe, or absent, X¹² is Val, Asp, Phe, or absent,X¹³ is Phe, Asp, or absent, X¹⁴ is Phe, Tyr, or absent, X¹⁵ is Asp orabsent, X¹⁶ is Tyr or absent; a LCDR1 sequence comprising the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 389), wherein X¹ isGln, X² is Gly, Ser, or Asp, X³ is Ile or Val, X⁴ is Ser, Leu, Asn, orGly, X⁵ is Asn, Tyr, Gly, or Ser, X⁶ is Tyr, Ser, Phe, or Trp, X⁷ is Seror absent, X⁸ is Asn or absent, X⁹ is Asn or absent, X¹⁰ is Lys orabsent, X¹¹ is Gln or absent, X¹² is Tyr or absent; a LCDR2 sequencecomprising the formula X¹-X²-X³ (SEQ ID NO: 390), wherein X¹ is Ala,Trp, Asp, Tyr, Lys, Gly, or Ser, X² is Ala or Thr, and X³ is Ser; and aLCDR3 sequence comprising the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ IDNO: 391), wherein X¹ is Gln, Leu, or His, X² is Lys, Gln, or His, X³ isTyr, Ser, or Leu, X⁴ is Tyr, Asn, Gly, Asp, or Ser, X⁵ is Ser, Asp, orAsn, X⁶ is Leu, Ala, Tyr, Thr, or Phe, X⁷ is Pro or Arg, X⁸ is Leu, Phe,Tyr, or Thr and X⁹ is Thr or absent.

In one embodiment, the invention features a human antibody orantigen-binding fragment specific for Fel d1, comprising a HCVR encodedby nucleotide sequence segments derived from V_(H), D_(H) and J_(H)germline sequences, and a LCVR encoded by nucleotide sequence segmentsderived from V_(K) and J_(K) germline sequences, with combinations asshown in Table 2.

The invention encompasses antibodies having a modified glycosylationpattern. In some applications, modification to remove undesirableglycosylation sites may be useful, or e.g., removal of a fucose moietyto increase antibody dependent cellular cytotoxicity (ADCC) function(see Shield et al. (2002) JBC 277:26733). In other applications,modification of galactosylation can be made in order to modifycomplement dependent cytotoxicity (CDC).

A second aspect provides an isolated antibody or antigen-bindingfragment thereof that competes for specific binding to Fel d1 with anantibody or antigen-binding fragment comprising the complementaritydetermining regions (CDRs) of a heavy chain variable region (HCVR),wherein the HCVR has an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162,178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370 and 460;and the CDRs of a light chain variable region (LCVR), wherein the LCVRhas an amino acid sequence selected from the group consisting of SEQ IDNOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218,234, 250, 266, 282, 298, 314, 330, 346, 362, 378 and 468.

One embodiment provides an isolated antibody or antigen-binding fragmentthereof that competes for specific binding to Fel d1 with an antibody orantigen-binding fragment comprising the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR), wherein the HCVRhas an amino acid sequence selected from the group consisting of SEQ IDNOs: 18, 66, 130, 162, 242, 306, 322, 370 and 460; and the CDRs of alight chain variable region (LCVR), wherein the LCVR has an amino acidsequence selected from the group consisting of SEQ ID NOs: 26, 74, 138,170, 250, 314, 330, 378 and 468.

In a related embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof that competes for specific binding toFel d1 with an antibody or antigen-binding fragment comprising the heavyand light chain CDRs contained within heavy and light chain sequencepairs selected from the group consisting of SEQ ID NOs: 18/26, 66/74,130/138, 162/170, 242/250, 306/314, 322/330, 370/378 and 460/468.

A third aspect provides an isolated antibody or antigen-binding fragmentthereof that binds the same epitope on Feld 1 as an antibody orantigen-binding fragment comprising the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR), wherein the HCVRhas an amino acid sequence selected from the group consisting of SEQ IDNOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226,242, 258, 274, 290, 306, 322, 338, 354, 370 and 460; and the CDRs of alight chain variable region (LCVR), wherein the LCVR has an amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282,298, 314, 330, 346, 362, 378 and 468.

One embodiment provides an isolated antibody or antigen-binding fragmentthereof that binds the same epitope on Fel d1 as an antibody orantigen-binding fragment comprising the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR), wherein the HCVRhas an amino acid sequence selected from the group consisting of SEQ IDNOs: 18, 66, 130, 162, 242, 306, 322, 370 and 460; and the CDRs of alight chain variable region (LCVR), wherein the LCVR has an amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282,298, 314, 330, 346, 362, 378 and 468.

In a related embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof that binds the same epitope on Fel d1as an antibody or antigen-binding fragment comprising the heavy andlight chain CDRs contained within heavy and light chain sequence pairsselected from the group consisting of SEQ ID NOs: 18/26, 66/74, 130/138,162/170, 242/250, 306/314, 322/330, 370/378 and 460/468.

A fourth aspect provides for a bi-specific antigen-binding molecule thatspecifically binds Fel d1, which comprises two antigen-binding domains(two arms) that comprise an HCVR amino acid sequence and a LCVR aminoacid sequence from any two or more antibodies described herein.

In one embodiment, the bi-specific antigen-binding molecule comprises afirst antigen-binding domain that comprises a HCVR amino acid sequenceas set forth in SEQ ID NO: 370 and a LCVR amino acid sequence as setforth in SEQ ID NO: 378, and a second antigen-binding domain thatcomprises a HCVR amino acid sequence as set forth in SEQ ID NO: 18 and aLCVR amino acid sequence as set forth in SEQ ID NO: 378.

In one embodiment, the bi-specific antigen-binding molecule comprises afirst antigen-binding domain that comprises three heavy chaincomplementarity determining regions (HCDR1, HCDR2 and HCDR3) consistingof the amino acid sequences as set forth in SEQ ID NOs: 372, 374 and376, respectively, and three light chain complementarity determiningregions (LCDR1, LCDR2 and LCDR3) consisting of the amino acid sequencesas set forth in SEQ ID NOs: 380, 382 and 384, respectively; and whereinthe second antigen-binding domain comprises three heavy chaincomplementarity determining regions (HCDR1, HCDR2 and HCDR3) consistingof the amino acid sequences as set forth in SEQ ID NOs: 20, 22 and 24,respectively, and three light chain complementarity determining regions(LCDR1, LCDR2 and LCDR3) consisting of the amino acid sequences as setforth in SEQ ID NOs: 380, 382 and 384, respectively.

In one embodiment, the bi-specific antigen-binding molecule comprises afirst antigen-binding domain that comprises a HCVR amino acid sequenceas set forth in SEQ ID NO: 306 and a LCVR amino acid sequence as setforth in SEQ ID NO: 314, and a second antigen-binding domain thatcomprises a HCVR amino acid sequence as set forth in SEQ ID NO: 18 and aLCVR amino acid sequence as set forth in SEQ ID NO: 314.

In one embodiment, the bi-specific antigen-binding molecule comprisesthree heavy chain complementarity determining regions (HCDR1, HCDR2 andHCDR3) consisting of the amino acid sequences as set forth in SEQ IDNOs: 308, 310 and 312, respectively, and three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) consistingof the amino acid sequences as set forth in SEQ ID NOs: 316, 318 and320, respectively; and wherein the second antigen-binding domaincomprises three heavy chain complementarity determining regions (HCDR1,HCDR2 and HCDR3) consisting of the amino acid sequences as set forth inSEQ ID NOs: 20, 22 and 24, respectively, and three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) consistingof the amino acid sequences as set forth in SEQ ID NOs: 316, 318 and320, respectively.

In one embodiment, the invention provides for an isolated antibodyspecific for Fel d1, or an antigen-binding fragment thereof thatcompetes for binding to Fel d1 with any one of the bi-specificantigen-binding molecules of the invention.

In one embodiment, the invention provides for an isolated antibodyspecific for Fel d1, or an antigen-binding fragment thereof that bindsto the same epitope on Fel d1 as any of the bi-specific antigen-bindingmolecules of the invention.

In one embodiment, the bi-specific antigen-binding molecule is anisolated human monoclonal antibody that binds specifically to Fel d1.

In one embodiment, the bi-specific antigen-binding molecule is anisolated human monoclonal antibody that binds specifically to Fel d1,wherein the human monoclonal antibody is a mono-specific antibody or abi-specific antibody.

In one embodiment, the invention provides for a pharmaceuticalcomposition comprising at least one bi-specific antigen-binding moleculeas described herein and a pharmaceutically acceptable carrier ordiluent.

In one embodiment, the invention provides for a method for treating apatient who demonstrates a sensitivity to, or an allergic reactionagainst, a cat, cat dander, cat hair or an extract thereof, or to Fel d1protein, or for treating at least one symptom or complication associatedwith a sensitivity to, or an allergic reaction against, a cat, catdander, cat hair or an extract thereof, or to Fel d1 protein, comprisingadministering an effective amount of one or more of the bi-specificantigen-binding molecules of the invention, or a pharmaceuticalcomposition comprising an effective amount of one or more of thebi-specific antigen-binding molecules of the invention, to a patient inneed thereof, wherein the patient demonstrates a reduced sensitivity to,or a diminished allergic reaction against a cat, cat dander, cat hair oran extract thereof, or to Fel d1 protein, or does not experience anysensitivity to, or allergic reaction to a cat, cat dander, cat hair oran extract thereof, or to Fel d1 protein, or wherein the patientdemonstrates a reduction in at least one symptom or complicationassociated with a sensitivity to, or an allergic reaction against, acat, cat dander, cat hair or an extract thereof, or to Fel d1 protein,or a reduction in the frequency and/or duration of at least one symptomor complication associated with a sensitivity to, or an allergicreaction against, a cat, cat dander, cat hair or an extract thereof, orto Fel d1 protein following administration of the bi-specificantigen-binding molecules or a composition comprising the bi-specificantigen-binding molecules of the invention.

In one embodiment, the invention provides for administering an effectiveamount of a second therapeutic agent along with at least one bi-specificantigen-binding molecule of the invention useful for diminishing anallergic reaction to a cat, cat dander, or to Fel d1 protein. The secondtherapeutic agent may be selected from the group consisting of acorticosteroid, a bronchial dilator, an antihistamine, epinephrine, adecongestant, a corticosteroid, another different antibody to Fel d1 anda peptide vaccine.

In one embodiment, the treatment with one or more bi-specificantigen-binding molecules of the invention alone, or in combination witha second therapeutic agent, may result in a reduction in allergicrhinitis, allergic conjunctivitis, allergic asthma, or an anaphylacticresponse following exposure of the patient to a cat, cat dander or toFel d1 protein.

In a fifth aspect, the invention provides nucleic acid moleculesencoding Fel d1 antibodies or fragments thereof. Recombinant expressionvectors carrying the nucleic acids of the invention, and host cells intowhich such vectors have been introduced, are also encompassed by theinvention, as are methods of producing the antibodies by culturing thehost cells under conditions permitting production of the antibodies, andrecovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113,129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337,353, 369 and 459, or a substantially identical sequence having at least90%, at least 95%, at least 98%, or at least 99% homology thereof.

In one embodiment, the HCVR is encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 17, 65, 129, 161, 241,305, 321, 369 and 459.

In one embodiment, the antibody or fragment thereof further comprises aLCVR encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169,185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361, 377 and 467or a substantially identical sequence having at least 90%, at least 95%,at least 98%, or at least 99% homology thereof.

In one embodiment, the LCVR is encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 25, 73, 137, 169, 249,313, 329, 377 and 467.

In one embodiment, the invention also provides an antibody orantigen-binding fragment of an antibody comprising a HCDR3 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199,215, 231, 247, 263, 279, 295, 311, 327, 343, 359, 375 and 465, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a LCDR3 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207,223, 239, 255, 271, 287, 303, 319, 335, 351, 367, 383 and 473, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

In one embodiment, the invention provides an antibody or fragmentthereof further comprising a HCDR1 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51,67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291,307, 323, 339, 355, 371 and 461, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; a HCDR2 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 5, 21, 37, 53, 69, 85,101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309,325, 341, 357, 373 and 463, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a LCDR1 domain encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123,139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347,363, 379 and 469, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;and a LCDR2 domain encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141,157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 333, 349, 365,381 and 471, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity.

A sixth aspect provides a pharmaceutical composition comprising atherapeutically effective amount of one or more isolated humanantibodies or antigen-binding fragments thereof that specifically bindFel d1, together with one or more pharmaceutically acceptableexcipients.

In one embodiment, the pharmaceutical composition comprises atherapeutically effective amount of two or more isolated humanantibodies or antigen-binding fragments thereof that specifically bindFel d1 together with one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises:

a) an isolated first fully human monoclonal antibody, or antigen-bindingfragment thereof that specifically binds Fel d1, which comprises a HCVRhaving an amino acid sequence as set forth is SEQ ID NO: 18; and a LCVRhaving an amino acid sequence as set forth is SEQ ID NO: 26; and

b) an isolated second fully human monoclonal antibody, orantigen-binding fragment thereof that specifically binds Fel d1, whichcomprises a HCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 66, 130, 162, 306, 322, 370 and 460; and aLCVR having an amino acid sequence selected from the group consisting ofSEQ ID NOs: 74, 138, 170, 314, 330, 378 and 468.

In one embodiment, the pharmaceutical composition comprises:

a) an isolated first fully human monoclonal antibody, or antigen-bindingfragment thereof that specifically binds Fel d1, which comprises a HCVRhaving an amino acid sequence as set forth is SEQ ID NO: 242; and a LCVRhaving an amino acid sequence as set forth is SEQ ID NO: 250; and

b) an isolated second fully human monoclonal antibody, orantigen-binding fragment thereof that specifically binds Fel d1, whichcomprises a HCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 306, 322 and 460; and a LCVR having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 314, 330and 468.

In one embodiment, the pharmaceutical composition comprises:

a) an isolated first fully human monoclonal antibody or antigen-bindingfragment thereof that specifically binds Fel d1, comprising a HCVR/LCVRamino acid sequence pair consisting of SEQ ID NOs: 18/26; and

b) an isolated second fully human monoclonal antibody or antigen-bindingfragment thereof that specifically binds Fel d1, comprising a HCVR/LCVRamino acid sequence pair selected from the group consisting of SEQ IDNOs: 66/74, 130/138, 162/170, 306/314, 322/330 370/378 and 460/468.

In one embodiment, the pharmaceutical composition comprises:

a) an isolated first human monoclonal antibody or antigen-bindingfragment thereof that binds specifically to Fel d1, comprising aHCVR/LCVR amino acid sequence pair consisting of SEQ ID NOs: 18/26; and

b) an isolated second human monoclonal antibody or antigen-bindingfragment thereof that binds specifically to Fel d1, comprising aHCVR/LCVR amino acid sequence pair consisting of SEQ ID NOs: 130/138.

In one embodiment, the pharmaceutical composition comprises:

a) an isolated first human monoclonal antibody or antigen-bindingfragment thereof that binds specifically to Fel d1, comprising aHCVR/LCVR amino acid sequence pair consisting of SEQ ID NOs: 18/26; and

b) an isolated second human monoclonal antibody or antigen-bindingfragment thereof that binds specifically to Fel d1, comprising aHCVR/LCVR amino acid sequence pair consisting of SEQ ID NOs: 322/330.

In one embodiment, the pharmaceutical composition comprises:

-   -   a) an isolated first human monoclonal antibody or        antigen-binding fragment thereof that binds specifically to Fel        d1, comprising a HCVR/LCVR amino acid sequence pair consisting        of SEQ ID NOs: 18/26; and    -   b) an isolated second human monoclonal antibody or        antigen-binding fragment thereof that binds specifically to Fel        d1, comprising a HCVR/LCVR amino acid sequence pair consisting        of SEQ ID NOs: 306/314.

In one embodiment, the pharmaceutical composition comprises:

a) an isolated first human monoclonal antibody or antigen-bindingfragment thereof that binds specifically to Fel d1, comprising aHCVR/LCVR amino acid sequence pair consisting of SEQ ID NOs: 18/26; and

-   -   b) an isolated second human monoclonal antibody or        antigen-binding fragment thereof that binds specifically to Fel        d1, comprising a HCVR/LCVR amino acid sequence pair consisting        of SEQ ID NOs: 370/378.

In one embodiment, the pharmaceutical composition comprises:

a) an isolated first fully human monoclonal antibody or antigen-bindingfragment thereof that specifically binds Fel d1, comprising a HCVR/LCVRamino acid sequence pair consisting of SEQ ID NOs: 242/250; and

-   -   b) an isolated second fully human monoclonal antibody or        antigen-binding fragment thereof that specifically binds Fel d1,        comprising a HCVR/LCVR amino acid sequence pair selected from        the group consisting of SEQ ID NOs: 306/314 and 322/330.

In one embodiment, the pharmaceutical composition comprises

-   -   a) an isolated first human monoclonal antibody or        antigen-binding fragment thereof that binds specifically to Fel        d1, comprising a HCVR/LCVR amino acid sequence pair consisting        of SEQ ID NOs: 242/250; and    -   b) an isolated second human monoclonal antibody or        antigen-binding fragment thereof that binds specifically to Fel        d1, comprising a HCVR/LCVR amino acid sequence pair consisting        of SEQ ID NOs: 306/314.

In one embodiment, the pharmaceutical composition comprises

-   -   a) an isolated first human monoclonal antibody or        antigen-binding fragment thereof that binds specifically to Fel        d1, comprising a HCVR/LCVR amino acid sequence pair consisting        of SEQ ID NOs: 242/250; and    -   b) an isolated second human monoclonal antibody or        antigen-binding fragment thereof that binds specifically to Fel        d1, comprising a HCVR/LCVR amino acid sequence pair consisting        of SEQ ID NOs: 322/330.

In one embodiment, the pharmaceutical composition comprises two or moreisolated human monoclonal antibodies that bind specifically to Fel d1,or antigen-binding fragments thereof, comprising HCVR/LCVR amino acidsequence pairs selected from the group consisting of SEQ ID NOs: 18/26,66/74, 130/138, 162/170, 242/250, 306/314, 322/330, 370/378 and 460/468.

In one embodiment, the pharmaceutical composition comprises fourisolated human monoclonal antibodies that bind specifically to Fel d1,or antigen-binding fragments thereof, wherein the human antibodies orantigen-binding fragments thereof comprise the HCVR/LCVR amino acidsequence pairs of SEQ ID NOs: 18/26, 66/74, 130/138 and 162/170.

In one embodiment, the invention features a composition, which is acombination of a therapeutically effective amount of one or moreanti-Fel d1 antibodies or antigen-binding fragments thereof of theinvention, and a therapeutically effective amount of a secondtherapeutic agent.

The second therapeutic agent may be a small molecule drug, aprotein/polypeptide, an antibody, a nucleic acid molecule, such as ananti-sense molecule, or a siRNA. The second therapeutic agent may besynthetic or naturally derived.

The second therapeutic agent may be any agent that is advantageouslycombined with an antibody or fragment thereof of the invention, forexample, a second antibody other than those described herein that iscapable of blocking the binding of Fel d1 to IgE present on mast cellsor basophils. A second therapeutic agent may also be any agent that isused as standard of care in treating an allergic response to anyallergen. Such second therapeutic agent may be an antihistamine,epinephrine, a decongestant, a corticosteroid, or a peptide vaccine.

In certain embodiments, the second therapeutic agent may be an agentthat helps to counteract or reduce any possible side effect(s)associated with the antibody or antigen-binding fragment of an antibodyof the invention, if such side effect(s) should occur.

It will also be appreciated that the antibodies and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the antibodies and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an antibody may be administered concurrently withanother agent used to treat the same disorder), or they may achievedifferent effects (e.g., control of any adverse effects). As usedherein, additional therapeutic agents that are normally administered totreat or prevent a particular disease, or condition, are appropriate forthe disease, or condition, being treated.

When multiple therapeutics are co-administered, dosages may be adjustedaccordingly, as is recognized in the pertinent art.

A seventh aspect provides a method for treating a patient whodemonstrates a sensitivity to, or an allergic reaction against, a cat,cat dander, cat hair extract, or to Fel d1 protein, or for treating atleast one symptom or complication associated with a sensitivity to, orallergic reaction against a cat, cat dander, cat hair extract, or to Feld1 protein, comprising administering an effective amount of one or moreisolated human monoclonal antibodies or antigen-binding fragmentsthereof that bind specifically to Fel d1, or a pharmaceuticalcomposition comprising an effective amount of one or more isolated humanmonoclonal antibodies or fragments thereof that binds specifically toFel d1, or an effective amount of one or more of the bi-specificantigen-binding molecules that specifically binds Fel d1, or apharmaceutical composition comprising an effective amount of one or moreof the bi-specific antigen-binding molecules that specifically binds toFel d1, to a patient in need thereof, wherein the sensitivity to, or anallergic reaction against, a cat, cat dander, cat hair extract, or toFel d1 protein is either prevented, or lessened in severity and/orduration, or at least one symptom or complication associated with thesensitivity to, or allergic reaction against, a cat, cat dander, cathair extract, or to Fel d1 protein is prevented, or ameliorated, or thatthe frequency and/or duration of, or the severity of the sensitivity toor allergic reaction against, a cat, cat dander, cat hair extract, or toFel d1 protein is reduced following administration of one or more of theisolated human monoclonal antibodies or fragments thereof that bindspecifically to Fel d1, or following administration of one or more ofthe bi-specific antigen-binding molecules that specifically binds Feld1, or following administration of a composition comprising any one ormore of the foregoing antibodies or bi-specific antigen-bindingmolecules.

In one embodiment, the invention provides a pharmaceutical compositioncomprising one or more of the antibodies of the invention, or one ormore of the bi-specific antigen-binding molecules that bindsspecifically to Fel d1 for use in treating a patient who demonstrates asensitivity to, or an allergic reaction against, a cat, cat dander, cathair extract, or to Fel d1 protein, or for treating at least one symptomor complication associated with a sensitivity to, or allergic reactionagainst a cat, cat dander, cat hair extract, or to Fel d1 protein,wherein the sensitivity to, or an allergic reaction against, a cat, catdander, cat hair extract, or to Fel d1 protein is either prevented, orlessened in severity and/or duration, or at least one symptom orcomplication associated with the sensitivity to, or allergic reactionagainst, a cat, cat dander, cat hair extract, or to Fel d1 protein isprevented, or ameliorated, or that the frequency and/or duration of, orthe severity of the sensitivity to or allergic reaction against, a cat,cat dander, cat hair extract, or to Fel d1 protein is reduced.

In one embodiment, the invention provides for use of a pharmaceuticalcomposition comprising one or more of the antibodies of the invention,or one or more of the bi-specific antigen-binding molecules that bindsspecifically to Fel d1 in the manufacture of a medicament for use intreating a patient who demonstrates a sensitivity to, or an allergicreaction against, a cat, cat dander, cat hair extract, or to Fel d1protein, or for treating at least one symptom or complication associatedwith a sensitivity to, or allergic reaction against a cat, cat dander,cat hair extract, or to Fel d1 protein, wherein the sensitivity to, oran allergic reaction against, a cat, cat dander, cat hair extract, or toFel d1 protein is either prevented, or lessened in severity and/orduration, or at least one symptom or complication associated with thesensitivity to, or allergic reaction against, a cat, cat dander, cathair extract, or to Fel d1 protein is prevented, or ameliorated, or thatthe frequency and/or duration of, or the severity of the sensitivity toor allergic reaction against, a cat, cat dander, cat hair extract, or toFel d1 protein is reduced.

In one embodiment, the invention provides use of a pharmaceuticalcomposition as described above, wherein the composition is administeredin combination with a second therapeutic agent useful for diminishing anallergic reaction to a cat, cat dander, cat hair extract, or to Fel d1protein. In one embodiment, the invention provides for use of thepharmaceutical composition as described above, wherein the secondtherapeutic agent is selected from a corticosteroid, a bronchialdilator, an antihistamine, epinephrine, a decongestant, anotherdifferent antibody to Fel d1 and a peptide vaccine.

In certain embodiments, the antibodies of the invention, or thebi-specific antigen-binding molecules that bind specifically to Fel d1may be capable of reducing, minimizing, or preventing at least onesymptom in a patient sensitive to the Fel d1 cat allergen, such assneezing, congestion, nasal blockage, coughing, wheezing,bronchoconstriction, rhinitis, or conjunctivitis.

In one embodiment, the antibodies of the invention, or the bi-specificantigen-binding molecules that bind specifically to Fel d1, or acomposition comprising one or more antibodies of the invention or one ormore of the antigen-binding molecules that bind specifically to Fel d1may be used to prevent more serious in vivo complications associatedwith an allergy to Fel d1, including asthmatic responses, anaphylacticshock, or even death resulting from anaphylaxis.

In one embodiment, the pharmaceutical composition is administered to thepatient in combination with a second therapeutic agent.

In another embodiment, the second therapeutic agent is selected from thegroup consisting of an antihistamine, epinephrine, a decongestant, acorticosteroid, another different antibody to Fel d1, a peptide vaccineand any other palliative therapy useful for reducing the severity of theallergic reaction or for ameliorating at least one symptom associatedwith the allergic reaction.

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference intheir entirety.

Definitions

The term “Fel d1” or “FELD1”, as used herein, refers to at least one Feld1 protein, either in natural/native form, or recombinantly produced.The Fel d1 protein comprises, or alternatively consists of, chain 1(also referred to as chain A) of Fel d1 (SEQ ID NO: 392) and chain 2(also referred to as chain B) of Fel d1 (SEQ ID NO: 393). The naturalFel d1 protein is an approximately 18 kDa heterodimeric glycoproteincomposed of two chains derived from two independent genes (See Duffort,O. A. et al., (1991), Mol. Immunol. 28:301-309; Kristensen, A. K. etal., (1997), Biol. Chem. 378:899-908; Kaiser L. et al. (2003), J. Biol.Chem. 278(39):37730-37735). A recombinantly produced Fel d1 protein isalso shown as SEQ ID NO: 396, wherein this sequence contains amino acidresidues 18 through 109 of Fel d1 chain B from GenBank accession numberNP_001041619.1 (without the signal sequence) fused in line with aminoacid residues 19-88 of chain A of Fel d1 from GenBank accession numberNP_001041618.1 (without the signal sequence and with a D27G mutation,which corresponds to the glycine at position 101 of SEQ ID NO: 396).Other recombinantly produced Fel d1 constructs of the invention areexemplified in SEQ ID NOs: 385, 394, 395 and 397.

“Chain 1”, or “chain A” of Fel d1 is a polypeptide comprising, oralternatively consisting of, an amino acid sequence of SEQ ID NO: 392,or a homologous sequence thereof. The term homologous sequence of SEQ IDNO:392, as used herein, refers to a polypeptide that has an identity toSEQ ID NO:392 which is greater than 70%, preferably greater than 80%,more preferably greater than 90%, and even more preferably greater than95%. The amino acid sequence of chain 1 of Fel d1 is also provided inGenBank as accession number P30438, or as accession numberNP_001041618.1, which also include the signal peptide which is removedin the mature protein.

“Chain 2”, or “chain B” of Fel d1 is a polypeptide comprising, oralternatively consisting of, an amino acid sequence of SEQ ID NO: 393,or a homologous sequence thereof. The term homologous sequence of SEQ IDNO: 393, as used herein, refers to a polypeptide that has an identity toSEQ ID NO:393 which is greater than 70%, preferably greater than 80%,more preferably greater than 90%, and even more preferably greater than95%. The amino acid sequence of chain 2 of Fel d1 is also provided inGenBank as accession number P30440, or as accession numberNP_001041619.1, which include the signal peptide which is removed in themature protein.

The term “Fel d1 fragment” as used herein, refers to a polypeptidecomprising or alternatively consisting of, at least one antigenic siteof Fel d1. In one embodiment, the term “Fel d1 fragment” as used herein,refers to a polypeptide comprising or alternatively consisting of atleast two antigenic sites of Fel d1. In one embodiment, the antigenicsites are covalently linked. In one embodiment, the antigenic sites arelinked by at least one peptide bond. In one embodiment, the twoantigenic sites are linked by at least one peptide bond and a spacerbetween the antigenic sites. In one embodiment, the at least twoantigenic sites derive from both chain 1 of Fel d1 and from chain 2 ofFel d1. In one embodiment, the at least two antigenic sites compriseamino acid sequences 23-92 of GenBank accession number P30438 and aminoacid sequences 18-109 of GenBank accession number P30440. In oneembodiment, the at least two antigenic sites derive from both chain 1 ofFel d1 and from chain 2 of Fel d1. In one embodiment, the at least twoantigenic sites comprise amino acid sequences 19-88 of GenBank accessionnumber NP_001041618.1 and amino acid sequences 18-109 of GenBankaccession number NP_001041619.1. In one embodiment, the at least twoantigenic sites comprise an amino acid sequence within any of SEQ IDNOs: 385, 394, 395, 396 or 397. In one embodiment, any of the Fel d1fragments are capable of inducing the production of antibodies in vivothat specifically bind to naturally occurring Fel d1, or torecombinantly produced Fel d1.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., Fel d1). The term “antibody”, as used herein, is intendedto refer to immunoglobulin molecules comprised of four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds (i.e., “full antibody molecules”), as well as multimersthereof (e.g. IgM) or antigen-binding fragments thereof. Each heavychain is comprised of a heavy chain variable region (“HCVR” or “V_(H)”)and a heavy chain constant region (comprised of domains C_(H)1, C_(H)2and C_(H)3). Each light chain is comprised of a light chain variableregion (“LCVR or “V_(L)”) and a light chain constant region (C_(L)). TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments ofthe invention, the FRs of the antibody (or antigen binding fragmentthereof) may be identical to the human germline sequences, or may benaturally or artificially modified. An amino acid consensus sequence maybe defined based on a side-by-side analysis of two or more CDRs.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. (2002), J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The fully human monoclonal antibodies that specifically bind to Fel d1,as disclosed herein, may comprise one or more amino acid substitutions,insertions and/or deletions in the framework and/or CDR regions of theheavy and light chain variable domains as compared to the correspondinggermline sequences. Such mutations can be readily ascertained bycomparing the amino acid sequences disclosed herein to germlinesequences available from, for example, public antibody sequencedatabases. The present invention includes antibodies, andantigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes fully human monoclonal antibodiescomprising variants of any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes antibodieshaving HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 orfewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences.

As used herein, the expression “antigen-binding molecule” means aprotein, polypeptide or molecular complex comprising or consisting of atleast one complementarity determining region (CDR) that alone, or incombination with one or more additional CDRs and/or framework regions(FRs), specifically binds to a particular antigen. In certainembodiments, an antigen-binding molecule is an antibody or a fragment ofan antibody, as those terms are defined elsewhere herein.

As used herein, the expression “bi-specific antigen-binding molecule”means a protein, polypeptide or molecular complex comprising at least afirst antigen-binding domain and a second antigen-binding domain (i.e.two arms). Each antigen-binding domain within the bi-specificantigen-binding molecule comprises at least one CDR that alone, or incombination with one or more additional CDRs and/or FRs, specificallybinds to a particular antigen. In the context of the present invention,the first antigen-binding domain specifically binds a first antigen onFel d1 and the second antigen-binding domain specifically binds asecond, distinct antigen on Fel d1.

The term “specifically binds,” or “binds specifically to”, or the like,means that an antibody or antigen-binding fragment thereof forms acomplex with an antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by an equilibriumdissociation constant of at least about 1×10⁻⁶ M or less (e.g., asmaller K_(D) denotes a tighter binding). Methods for determiningwhether two molecules specifically bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. As described herein, antibodies have been identified bysurface plasmon resonance, e.g., BIACORE™, which bind specifically toFel d1. Moreover, multi-specific antibodies that bind to Fel d1 and oneor more additional antigens or a bi-specific that binds to two differentregions of Fel d1 (for example, chain 1 and/or chain 2 of Fel d1) arenonetheless considered antibodies that “specifically bind”, as usedherein.

The term “high affinity” antibody refers to those mAbs having a bindingaffinity to Fel d1, expressed as K_(D), of at least 10⁻⁸ M; preferably10⁻⁹M; more preferably 10⁻¹⁰M, even more preferably 10⁻¹¹ M, even morepreferably 10⁻¹² M, as measured by surface plasmon resonance, e.g.,BIACORE™ or solution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antibody thatdissociates from Fel d1, with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

The terms “antigen-binding portion” of an antibody, “antigen-bindingfragment” of an antibody, and the like, as used herein, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. The terms “antigen-binding portion” of anantibody, or “antibody fragment”, as used herein, refers to one or morefragments of an antibody that retain the ability to bind to Fel d1.

The specific embodiments, antibody or antibody fragments of theinvention may be conjugated to a therapeutic moiety (“immunoconjugate”),such as a corticosteroid, a second anti-Fel d1 antibody, or epinephrine,a vaccine, or any other therapeutic moiety useful for treating anallergic response to Fel d1.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies (Abs) havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds Fel d1, or a fragment thereof, is substantially freeof Abs that specifically bind antigens other than Fel d1.

A “blocking antibody” or a “neutralizing antibody”, as used herein (oran “antibody that neutralizes Fel d1 activity”), is intended to refer toan antibody, or an antigen binding portion thereof, whose binding to Feld1 results in inhibition of at least one biological activity of Fel d1.For example, an antibody of the invention may aid in preventing theprimary allergic response to Fel d1. Alternatively, an antibody of theinvention may demonstrate the ability to prevent a secondary allergicresponse to Fel d1, or at least one symptom of an allergic response toFel d1, including sneezing, coughing, an asthmatic condition, or ananaphylactic response caused by Fel d1. This inhibition of thebiological activity of Fel d1 can be assessed by measuring one or moreindicators of Fel d1 biological activity by one or more of severalstandard in vitro or in vivo assays (such as a passive cutaneousanaphylaxis assay, as described herein) or other in vivo assays known inthe art (for example, other animal models to look at protection fromchallenge with Fel d1 following administration of one or more of theantibodies described herein).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. The term“epitope” also refers to a site on an antigen to which B and/or T cellsrespond. It also refers to a region of an antigen that is bound by anantibody. Epitopes may be either linear or conformational. A linearepitope is one produced by adjacent amino acid residues in a polypeptidechain. A conformational epitope is produced by spatially juxtaposedamino acids from different segments of the linear polypeptide chain. Incertain embodiments, epitopes may include determinants that arechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. Epitopes mayalso be defined as structural or functional. Functional epitopes aregenerally a subset of the structural epitopes and have those residuesthat directly contribute to the affinity of the interaction. Epitopesformed from contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, and more usually, at least 5 or 8-10amino acids in a unique spatial conformation.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below. A nucleic acid molecule havingsubstantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantiallysimilar amino acid sequence as the polypeptide encoded by the referencenucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positions,which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and (1997)Nucleic Acids Res. 25:3389 402, each of which is herein incorporated byreference.

In specific embodiments, the antibody or antibody fragment for use inthe method of the invention may be mono-specific, bi-specific, ormulti-specific. Multi-specific antibodies may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for epitopes of more than one target polypeptide. Anexemplary bi-specific antibody format that can be used in the context ofthe present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise an Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 mAbs;N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the caseof IgG2 mAbs; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 mAbs. Variations on the bi-specific antibody format describedabove are contemplated within the scope of the present invention.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

The antibodies of the invention may be used to “desensitize” acat-sensitive individual. The term to “desensitize” is defined herein asto decrease the allergic-reactivity of a cat-sensitive individual toexposure to cats, cat dander or products thereof, e.g. Fel d1 (to alevel less than that which the cat-sensitive individual would otherwiseexperience).

General Description

The domestic cat is a source of many indoor allergens and the severityof the symptoms in individuals who demonstrate a sensitivity to catallergens ranges from a relatively mild rhinitis and conjunctivitis to apotentially life-threatening asthmatic condition (Lau, S. et al. (2000),Lancet 356:1392-1397). While patients who demonstrate such a sensitivityto cats appear to be responsive to different molecules found in catdander and pelts, the major allergen appears to be Fel d1 (Felisdomesticus allergen 1). It has been shown that greater than 80% ofpatients who are allergic to cats have IgE antibodies to this allergen(van Ree, R. et al. (1999), J. Allergy Clin. Immunol 104:1223-1230).

The Fel d1 protein is an approximately 18 kDa heterodimeric acidicglycoprotein that contains about 10-20% of N-linked carbohydrates. Eachheterodimer comprises two polypeptide chains that are encoded by twoseparate genes (Duffort, O A, et al., (1991), Mol. Immunol. 28:301-309;Morgenstern, J P, et al., (1991), PNAS 88:9690-9694; Griffith, I. J., etal. (1992), Gene 113:263-268). Chain 1 comprises about 70 amino acidresidues and chain 2 comprises about 90-92 amino acid residues. Threeinterchain disulfide bonds linking the two chains in natural Fel d1 havebeen proposed (Kristensen, A. K. et al. (1997), Biol. Chem. 378:899-908)and confirmed for recombinant Fel d1 in the crystal structure (Kaiser,L. et al. (2003), J. Biol. Chem. 278:37730-37735; Kaiser, L. et al.,(2007), J. Mol. Biol. 370:714-727). Although each chain is sometimesindividually referred to as “Fel d 1”, both chains are needed for thefull protein allergen.

Fel d1 is produced by sebaceous glands, squamous glands and squamousepithelial cells and is transferred to the pelt by licking and grooming(Bartholome, K. et al. (1985), J. Allergy Clin. Immunol. 76:503-506;Charpin, C. et al. (1991), J. Allergy Clin. Immunol. 88:77-82;Dabrowski, A. J. (1990), et al. J. Allergy Clin. Immunol. 86:462-465).It is also present in the salivary, perianal and lachrymal glands(Andersen, M. C., et al. (1985), J. Allergy Clin. Immunol. 76:563-569;van Milligen, F. J. et al., (1992), Int. Arch. Allergy Appl. Immunol.92:375-378) and the principal reservoirs appear to be the skin and thefur (Mata, P. et al. (1992), Ann. Allergy 69(4):321-322).

The Fel d1 protein is of an unknown function to the animal but causes anIgG or IgE reaction in sensitive humans (either as an allergic orasthmatic response). Although other cat allergens are known, includingFel d2 (albumin) and Fel d3 (cystatin), 60% to 90% of the anti-cat IgEproduced is directed against Fel d1 (Leitermann, K. et al., (1984), JAllergy Clin. Immunol. 74:147-153; Lowenstein, H. et al., (1985),Allergy 40:430-441; van Ree, R. et al., (1999), J. Allergy Clin.Immunol. 104:1223-1230; Ichikawa, K. et al., (2011), Clin. Exp. Allergy,31:1279-1286).

Immunoglobulin E (IgE) is responsible for type 1 hypersensitivity, whichmanifests itself in allergic rhinitis, allergic conjunctivitis, hayfever, allergic asthma, bee venom allergy, and food allergies. IgEcirculates in the blood and binds to high-affinity Fc receptors for IgEon basophils and mast cells. In most allergic responses, the allergensenter the body through inhalation, ingestion, or through the skin. Theallergen then binds to preformed IgE already bound to the high affinityreceptor on the surfaces of mast cells and basophils, resulting incross-linking of several IgE molecules and triggering the release ofhistamine and other inflammatory mediators causing the various allergicsymptoms.

The treatment for cat allergies includes desensitization therapy, whichinvolves repeated injections with increasing dosages of either a crudecat dander extract, or short peptides derived from Fel d1. Using thecrude extract of cat dander, Lilja et. al. demonstrated that after threeyears of such treatment, patients allergic to cats still exhibitedsystemic symptoms (Lilja, Q. et al. (1989), J. Allergy Clin. Immunol.83:37-44 and Hedlin, et al. (1991), J. Allergy Clin. Immunol.87:955-964). Using short peptides derived from Fel d1 fordesensitization resulted in a non-significant difference between thepeptide group and the placebo control group (Oldfield, W. L. et al.,(2002), Lancet, 360:47-53). Efficacy was only observed when largeamounts (750 ug) of the short peptide were administered to patients(Norman, P. S. et al. (1996), Am. J. Respir. Crit. Care Med.154:1623-1628). Furthermore, asthmatic reactions have been reported inpatients given both crude extracts from cat dander, as well as inpatients given short Fel d1 peptide treatment. Accordingly, there is aneed in the field of cat allergy treatment for alternative strategiesfor treating patients sensitive to cat allergens, in particular Fel d1.

Antibodies have been proposed as a general treatment strategy forallergies, since they may be able to block the entry of allergenicmolecules into the mucosal tissues, or may bind the allergen before ithas the opportunity to bind to the IgE bound to the high affinityreceptor on mast cells or basophils, thus preventing the release ofhistamine and other inflammatory mediators from these cells. U.S. Pat.No. 5,670,626 describes the use of monoclonal antibodies for thetreatment of IgE-mediated allergic diseases such as allergic rhinitis,allergic asthma, and allergic conjunctivitis by blocking the binding ofallergens to the mucosal tissue. U.S. Pat. No. 6,849,259 describes theuse of allergen-specific antibodies to inhibit allergic inflammation inan in vivo mouse model of allergy. Milk-based and egg-based antibodysystems have been described. For example, US20030003133A1 disclosesusing milk as a carrier for allergens for inducing oral tolerance to catdander and other allergens. Compositions and methods for reducing anallergic response in an animal to an allergen in the environment throughuse of a molecule that inhibits the ability of the allergen to bind tomast cells was described in US2010/0143266. Other antibodies to Fel d1were described by de Groot et. al. (de Groot et. al., (1988), J. AllergyClin. Immunol. 82:778-786).

The fully human antibodies described herein demonstrate specific bindingto Fel d1 and may be useful for treating patients suffering from catallergies, in particular, in patients who demonstrate sensitivity to theFel d1 allergen. The use of such antibodies may be an effective means oftreating patients suffering from allergies to cat dander, or they may beused to prevent a heightened response to Fel d1 upon secondary exposure,or the accompanying symptoms associated with the allergy, or may be usedto lessen the severity and/or the duration of the allergic responseassociated with a primary exposure to a cat harboring the Fel d1allergen or with the recurrence of the symptoms upon secondary exposure.They may be used alone or as adjunct therapy with other therapeuticmoieties or modalities known in the art for treating such allergies,such as, but not limited to, treatment with corticosteroids orepinephrine. They may be used in conjunction with a second or thirddifferent antibody specific for Fel d1. They may be used withallergen-specific immunotherapy (SIT).

In certain embodiments, the antibodies of the invention are obtainedfrom mice immunized with a primary immunogen, such as natural Fel d1,which may be purchased commercially (See, for example, Indoor Biotech,#NA-FD1-2), or may be produced recombinantly. In certain embodiments,the immunogen may be either chain 1 of Fel d1, or chain 2 of Fel d1, ormay be a combination of both chain 1 and chain 2 administeredsequentially, or concurrently. The full-length amino acid sequence ofchain 1 (also referred to as FELD1 A) is shown as SEQ ID NO: 392.Full-length amino acid sequences for chain 1 may also be found inGenBank accession numbers P30438 and NP_001041618.1. The full-lengthamino acid sequence of chain 2 (also referred to as FELD1 B) is shown asSEQ ID NO: 393. Full-length amino acid sequences for chain 2 may also befound in GenBank accession numbers PP30440 and NP_001041619.1.

In certain embodiments, the recombinantly produced Fel d1 immunogen maybe made by direct fusion of the two chains of Fel d1, as described inKaiser et. al., to produce a fusion product that has a similar refoldingpattern to that of natural Fel d1 (Kaiser, L. et al., (2003), J. Biol.Chem. 278(39):37730-37735). In certain embodiments, the immunogen may bea fusion protein such as that shown in the constructs of SEQ ID NOs:385, 394, 395, 396 or 397, followed by immunization with a secondaryimmunogen, or with an immunogenically active fragment of the natural orrecombinantly produced Fel d1.

The immunogen may be a biologically active and/or immunogenic fragmentof natural or recombinantly produced Fel d1, or DNA encoding the activefragment thereof. The fragment may be derived from either the N-terminalor C-terminal of either chain 1 or chain 2, or from the N terminal orthe C terminal of both chain 1 and chain 2. Fragments may be obtainedfrom any site within chain 1 or chain 2 to be used as an immunogen forpreparing antibodies to Fel d1.

In certain embodiments, the immunogen may be a fusion protein comprisingany one or more of the following: i) amino acid residues 18-109 of chain2 of Fel d1 (See GenBank accession number P30440 and also SEQ ID NO:393) fused via the C terminus directly with the N terminus of amino acidresidues 23-92 of chain 1 of Fel d1 (See GenBank accession number P30438and also SEQ ID NO: 392); ii) amino acid residues 23-92 of chain 1 ofFel d1 (See GenBank accession number P30438 and also SEQ ID NO: 392)fused via the C terminus to the N terminus of amino acid residues 18-109of chain 2 of Fel d1 (See GenBank accession number P30440 and also SEQID NO: 393); iii) amino acid residues 18-109 of chain 2 of Fel d1 (SeeGenBank accession number NP_001041619.1) fused via the C terminusdirectly with the N terminus of amino acid residues 19-88 of chain 1 ofFel d1 (See GenBank accession number NP_001041618.1), such as theconstruct shown in SEQ ID NO: 394 or 396; iv) amino acid residues 19-88of chain 1 of Fel d1 (See GenBank accession number NP_001041618.1) fusedvia the C terminus to the N terminus of amino acid residues 18-109 ofchain 2 of Fel d1 (See GenBank accession number NP_001041619.1). Seealso SEQ ID NO: 395. In certain embodiments, the fusion protein may havea tag at the C terminal end of the construct, such as amyc-myc-hexahistidine tag (See SEQ ID NOs: 385, 396 or 397 for suchconstructs.). In related embodiments, the fusion protein may have amouse antibody Fc region coupled at the C terminal end of the construct(See SEQ ID NOs: 394 or 395 for such constructs.). In certainembodiments, chains 1 and 2 are coupled via a linker known to thoseskilled in the art, e.g. (G₄S)₃ (See SEQ ID NOs: 395 and 397 for such aconstruct.).

In certain embodiments, antibodies that bind specifically to Fel d1 maybe prepared using fragments of the above-noted regions, or peptides thatextend beyond the designated regions by about 5 to about 20 amino acidresidues from either, or both, the N or C terminal ends of the regionsdescribed herein. In certain embodiments, any combination of theabove-noted regions or fragments thereof may be used in the preparationFel d1 specific antibodies. In certain embodiments, any one or more ofthe above-noted regions of Fel d1, or fragments thereof may be used forpreparing monospecific, bispecific, or multispecific antibodies.

Antigen-Binding Fragments of Antibodies

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. The terms “antigen-binding portion”of an antibody, or “antibody fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to either chain 1 and/or chain 2 of Fel d1. An antibody fragmentmay include a Fab fragment, a F(ab′)₂ fragment, a Fv fragment, a dAbfragment, a fragment containing a CDR, or an isolated CDR.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and (optionally) constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1;V_(H)-C_(H)2; V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2, (v)V_(H)-C_(H)1-C_(H)2-C_(H)3, (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemono-specific or multi-specific (e.g., bi-specific). A multi-specificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multi-specific antibody format, including theexemplary bi-specific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind to Feld1.

Using VELOCIMMUNE™ technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating monoclonal antibodies, high affinity chimericantibodies to Fel d1 are initially isolated having a human variableregion and a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired humanconstant region to generate the fully human antibody of the invention,for example wild-type or modified IgG1 or IgG4. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) of from about 10⁻¹² through about10⁻⁹ M, when measured by binding to antigen either immobilized on solidphase or in solution phase. The mouse constant regions are replaced withdesired human constant regions to generate the fully human antibodies ofthe invention. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Bioequivalents

The anti-Fel d1 antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies, but that retain the ability to bindFel d1. Such variant antibodies and antibody fragments comprise one ormore additions, deletions, or substitutions of amino acids when comparedto parent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, theantibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an antibody or antibody fragment that is essentiallybioequivalent to an antibody or antibody fragment of the invention.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of the antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include antibody variants comprising aminoacid changes, which modify the glycosylation characteristics of theantibodies, e.g., mutations that eliminate or remove glycosylation.

Biological Characteristics of the Antibodies

In general, the antibodies of the present invention may function bybinding to either chain 1 or to chain 2 of Fel d1, or to both chain 1and chain 2 of Fel d1 or to a fragment of either chain 1 or chain 2.

In certain embodiments, the antibodies of the present invention may bindto an epitope located in at least the C-terminal region of either chain1 or chain 2 of Fel d1. In one embodiment, the antibodies may bind to anepitope within the N-terminal region of either chain 1 or chain 2 of Feld1.

In certain embodiments, the antibodies of the present invention mayfunction by blocking or inhibiting the binding of IgE to mast cells orbasophils in a patient sensitive to the fel d1 allergen.

In certain embodiments, the antibodies of the present invention mayfunction by binding to any other region or fragment of the full lengthchain 1 or chain 2 of the natural Fel d1 protein, the amino acidsequence of which is shown in SEQ ID NO: 392 (chain 1) and SEQ ID NO:393 (chain 2).

In certain embodiments, the antibodies of the present invention may bebi-specific antibodies. The bi-specific antibodies of the invention maybind one epitope in chain 1 and may also bind one epitope in chain 2. Incertain embodiments, the bi-specific antibodies of the invention maybind two different epitopes in chain 1. In certain embodiments, thebi-specific antibodies of the invention may bind two different epitopesin chain 2. In certain embodiments, the bi-specific antibodies of theinvention may bind to two different sites within the same helix oneither one of chain 1 or chain 2, or may bind to the same helix on bothchain 1 and chain 2. The structure of Fel d1 is described in greaterdetail in Kaiser et. al. (Kaiser, L. et. al. (2003), J. Biol. Chem. 278(39):37730-37735), whereby the authors note that Fel d1 consists ofeight helices, H1-H4 and H5-H8, which correspond to chains 2 and 1,respectively, in natural Fel d1.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that binds to chain 1and/or chain 2 of Fel d1, wherein the antibody or fragment thereofexhibits one or more of the following characteristics: (i) comprises aHCVR having an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210,226, 242, 258, 274, 290, 306, 322, 338, 354 and 370, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (ii) comprises a LCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 10, 26,42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266,282, 298, 314, 330, 346, 362 and 378, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; (iii) comprises a HCDR3 domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 8,24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248,264, 280, 296, 312, 328, 344, 360 and 376, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a LCDR3 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 16, 32, 48,64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288,304, 320, 336, 352, 368 and 384, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; (iv) comprises a HCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52,68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292,308, 324, 340, 356 and 372, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a HCDR2 domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134,150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358 and374, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a LCDR1domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204,220, 236, 252, 268, 284, 300, 316, 332, 348, 364 and 380, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a LCDR2 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222,238, 254, 270, 286, 302, 318, 334, 350, 366 and 382, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (v) binds to chain 1 and/or chain 2of Fel d1 with a K_(D) equal to or less than 10⁻⁹; (vi) does notcross-react with, or bind to, uteroglobin; or (vii) blocks dyeextravasation in vivo in a passive cutaneous anaphylaxis (PCA) mousemodel using Fel d1 specific mouse IgE.

In one embodiment, the invention provides for the use of a combinationof two or more fully human antibodies of the invention, or fragmentsthereof, for preparation of a composition, wherein the antibodies bindto chain 1 and/or chain 2 of Fel d1, and wherein each antibody orfragment thereof contained within the composition exhibits one or moreof the following characteristics: (i) comprise a HCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258,274, 290, 306, 322, 338, 354 and 370, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; (ii) comprises a LCVR having an amino acidsequence selected from the group consisting of SEQ ID NO: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282,298, 314, 330, 346, 362 and 378, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; (iii) comprises a HCDR3 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56,72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296,312, 328, 344, 360 and 376, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a LCDR3 domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128,144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352,368 and 384, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; (iv)comprises a HCDR1 domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132,148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356 and372, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a HCDR2domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198,214, 230, 246, 262, 278, 294, 310, 326, 342, 358 and 374, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a LCDR1 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220,236, 252, 268, 284, 300, 316, 332, 348, 364 and 380, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a LCDR2 domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 14, 30,46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270,286, 302, 318, 334, 350, 366 and 382, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; (v) binds to chain 1 and/or chain 2 of Feld1 with a K_(D) equal to or less than 10⁻⁹; (vi) does not cross-reactwith, or bind to, uteroglobin; (vii) blocks dye extravasation in vivo ina passive cutaneous anaphylaxis (PCA) mouse model using Fel d1 specificmouse IgE; or (viii) when combined with a second antibody or antigenbinding fragment thereof of the invention, decreases the frequency ofmucous secreting cells in the lungs of Fel d1 challenged animals.

Certain Fel d1 antibodies of the present invention, when used alone, orin combination, are able to bind to and neutralize at least onebiological effect of Fel d1, as determined by in vitro or in vivoassays. The ability of the antibodies of the invention to bind to andneutralize the activity of Fel d1 may be measured using any standardmethod known to those skilled in the art, including binding assays, orneutralization of activity (e.g., protection from anaphylaxis) assays,as described herein.

Non-limiting, exemplary in vitro assays for measuring binding activityare illustrated in Examples 4, herein. In Examples 4, the bindingaffinities and kinetic constants of human anti-Fel d1 antibodies weredetermined by surface plasmon resonance and the measurements wereconducted on a T200 Biacore instrument.

The Fel d1 proteins or peptides may be modified to include addition orsubstitution of certain residues for tagging or for purposes ofconjugation to carrier molecules, such as, KLH. For example, a cysteinemay be added at either the N terminal or C terminal end of a peptide, ora linker sequence may be added to prepare the peptide for conjugationto, for example, KLH for immunization. The antibodies specific for Feld1 may contain no additional labels or moieties, or they may contain anN-terminal or C-terminal label or moiety. In one embodiment, the labelor moiety is biotin. In a binding assay, the location of a label (ifany) may determine the orientation of the peptide relative to thesurface upon which the peptide is bound. For example, if a surface iscoated with avidin, a peptide containing an N-terminal biotin will beoriented such that the C-terminal portion of the peptide will be distalto the surface.

Epitope Mapping and Related Technologies

The term “epitope,” as used herein, refers to an antigenic determinantthat interacts with a specific antigen binding site in the variableregion of an antibody molecule known as a paratope. A single antigen mayhave more than one epitope. Thus, different antibodies may bind todifferent areas on an antigen and may have different biological effects.Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain. Incertain circumstance, an epitope may include moieties of saccharides,phosphoryl groups, or sulfonyl groups on the antigen.

The present invention includes anti-Fel d1 antibodies which interactwith one or more amino acids found within one or more regions of chain 1or chain 2 of the Fel d1 molecule including, e.g., chain 1 (chain A) asshown in SEQ ID NO: 392, or chain 2 (chain B) as shown in SEQ ID NO:393, or within comparable regions of a recombinantly produced Fel d1protein, as shown in any one of SEQ ID NOs: 385, 394, 395, 396 or 397.The epitope to which the antibodies bind may consist of a singlecontiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within anyof the aforementioned regions or segments of the Fel d1 molecule (e.g. alinear epitope in either chain 1 or chain 2, or in a region that spansboth chain 1 and chain 2). Alternatively, the epitope may consist of aplurality of non-contiguous amino acids (or amino acid sequences)located within either or both of the aforementioned regions or segmentsof the Fel d1 molecule (e.g. a conformational epitope).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,for example, routine cross-blocking assays, such as that described inAntibodies, Harlow and Lane (Cold Spring Harbor Press, Cold SpringHarb., N.Y.). Other methods include alanine scanning mutationalanalysis, peptide blot analysis (Reineke (2004) Methods Mol Biol248:443-63), peptide cleavage analysis crystallographic studies and NMRanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Protein Science 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water and exchangeable protons within aminoacids that are protected by the antibody complex undergodeuterium-to-hydrogen back-exchange at a slower rate than exchangeableprotons within amino acids that are not part of the interface. As aresult, amino acids that form part of the protein/antibody interface mayretain deuterium and therefore exhibit relatively higher mass comparedto amino acids not included in the interface. After dissociation of theantibody, the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-raycrystallography of the antigen/antibody complex may also be used forepitope mapping purposes.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (US2004/0101920, herein specifically incorporated by reference in itsentirety). Each category may reflect a unique epitope either distinctlydifferent from or partially overlapping with epitope represented byanother category. This technology allows rapid filtering of geneticallyidentical antibodies, such that characterization can be focused ongenetically distinct antibodies. When applied to hybridoma screening,MAP may facilitate identification of rare hybridoma clones that producemAbs having the desired characteristics. MAP may be used to sort theantibodies of the invention into groups of antibodies binding differentepitopes.

In certain embodiments, the anti-Fel d1 antibodies or antigen-bindingfragments thereof bind an epitope within any one or more of the regionsexemplified in chain 1 or chain 2 of Fel d1, either in natural form, asexemplified in SEQ ID NO: 392 (chain 1) and SEQ ID NO: 393 (chain 2), orrecombinantly produced, as exemplified in any of SEQ ID NOS: 385, 394,395, 396, and 397, or to a fragment thereof. In certain embodiments, theantibodies of the invention, as shown in Table 1, interact with at leastone amino acid sequence selected from the group consisting of amino acidresidues ranging from about position 15 to about position 24 of SEQ IDNO: 396; amino acid residues ranging from about position 85 to aboutposition 103 of SEQ ID NO: 396; amino acid residues ranging from aboutposition 85 to about position 104 of SEQ ID NO: 396; amino acid residuesranging from about position 113 to about position 116 of SEQ ID NO: 396.These regions are further exemplified in SEQ ID NOs: 402, 403, 404, 412and 426.

The present invention also includes anti-Fel d1 antibodies that bind tothe same epitope, or a portion of the epitope, as any of the specificexemplary antibodies described herein in Table 1, or an antibody havingthe CDR sequences of any of the exemplary antibodies described inTable 1. Likewise, the present invention also includes anti-Fel d1antibodies that compete for binding to Fel d1 or a Fel d1 fragment withany of the specific exemplary antibodies described herein in Table 1, oran antibody having the CDR sequences of any of the exemplary antibodiesdescribed in Table 1.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-Fel d1 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-Fel d1antibody of the invention, the reference antibody is allowed to bind toa Fel d1 protein or peptide under saturating conditions. Next, theability of a test antibody to bind to the Fel d1 molecule is assessed.If the test antibody is able to bind to Fel d1 following saturationbinding with the reference anti-Fel d1 antibody, it can be concludedthat the test antibody binds to a different epitope than the referenceanti-Fel d1 antibody. On the other hand, if the test antibody is notable to bind to the Fel d1 molecule following saturation binding withthe reference anti-Fel d1 antibody, then the test antibody may bind tothe same epitope as the epitope bound by the reference anti-Fel d1antibody of the invention.

To determine if an antibody competes for binding with a referenceanti-Fel d1 antibody, the above-described binding methodology isperformed in two orientations: In a first orientation, the referenceantibody is allowed to bind to a Fel d1 molecule under saturatingconditions followed by assessment of binding of the test antibody to theFel d1 molecule. In a second orientation, the test antibody is allowedto bind to a Fel d1 molecule under saturating conditions followed byassessment of binding of the reference antibody to the Fel d1 molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the Fel d1 molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to Fel d1.As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the identical epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

Immunoconjugates

The invention encompasses a human anti-Fel d1 monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as an agentthat is capable of reducing the severity of an allergic response to theFel d1 allergen present in cat dander or on cats, or in an area of theenvironment where cats may reside, or to ameliorate at least one symptomassociated with exposure to cats, cat dander or to the Fel d1 allergen,including rhinitis, conjunctivitis, or breathing difficulties, or theseverity thereof. Such an agent may be a corticosteroid, a seconddifferent antibody to Fel d1, or a vaccine. The type of therapeuticmoiety that may be conjugated to the Fel d1 antibody will take intoaccount the condition to be treated and the desired therapeutic effectto be achieved. Alternatively, if the desired therapeutic effect is totreat the sequelae or symptoms associated with exposure to the Fel d1allergen, or any other condition resulting from such exposure, such as,but not limited to, rhinitis or conjunctivitis, it may be advantageousto conjugate an agent appropriate to treat the sequelae or symptoms ofthe condition, or to alleviate any side effects of the antibodies of theinvention. Examples of suitable agents for forming immunoconjugates areknown in the art, see for example, WO 05/103081.

Multi-Specific Antibodies

The antibodies of the present invention may be mono-specific,bi-specific, or multi-specific. Multi-specific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The antibodies of the present inventioncan be linked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multi-specific antibody with a second bindingspecificity. For example, the present invention includes bi-specificantibodies wherein one arm of an immunoglobulin may be specific forchain 1 of Fel d1, or a fragment thereof, and the other arm of theimmunoglobulin may be specific for chain 2 of Fel d1, or a secondtherapeutic target, or may be conjugated to a therapeutic moiety.

Certain exemplary embodiments of the present invention include abi-specific antigen-binding molecule, which is a bi-specific antibody.Each antigen-binding domain of a bi-specific antibody comprises a heavychain variable domain (HCVR) and a light chain variable domain (LCVR).The HCVR may also be referred to as a V_(H) region, and the LCVR mayalso be referred to as a V_(L) region. Typically, each HCVR and LCVRcomprises three CDRs interspersed with four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The three CDRs within an HCVR may be referredto herein as HCDR1, HCDR2 and HCDR3; while the three CDRs within an LCVRmay be referred to herein as LCDR1, LCDR2 and LCDR3.

In the bi-specific antigen-binding molecules of the present invention,each antigen-binding domain may comprise or consist of a full antibodymolecule or an antigen-binding fragment of an antibody. The terms“antigen-binding portion” of an antibody, “antigen-binding fragment” ofan antibody, and the like, as used herein, include any naturallyoccurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. Antigen-binding fragments of an antibody maybe derived, e.g., from full antibody molecules using any suitablestandard techniques such as proteolytic digestion or recombinant geneticengineering techniques involving the manipulation and expression of DNAencoding antibody variable and optionally constant domains. Such DNA isknown and/or is readily available from, e.g., commercial sources, DNAlibraries (including, e.g., phage-antibody libraries), or can besynthesized. The DNA may be sequenced and manipulated chemically or byusing molecular biology techniques, for example, to arrange one or morevariable and/or constant domains into a suitable configuration, or tointroduce codons, create cysteine residues, modify, add or delete aminoacids, etc.

Non-limiting examples of antigen-binding fragments that may be includedin the bi-specific antigen-binding molecules of the present inventioninclude: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments;(iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAbfragments; and (vii) minimal recognition units consisting of the aminoacid residues that mimic the hypervariable region of an antibody. Otherengineered molecules, such as domain-specific antibodies, single domainantibodies, domain-deleted antibodies, chimeric antibodies, CDR-graftedantibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies(e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of a bi-specificantigen-binding molecule may contain at least one variable domaincovalently linked to at least one constant domain. Non-limiting,exemplary configurations of variable and constant domains that may befound within an antigen-binding domain of a bi-specific antigen-bindingmolecule may include: (i) V_(H)-C_(H)1; (ii) V_(H)-C_(H)2; (iii)V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v) V_(H)-C_(H)1-C_(H)2-C_(H)3;(vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L); V_(L)-C_(H)1; (ix)V_(L)-C_(H)2; (X) V_(L)-C_(H)3; (xi) V_(L)-C_(H)1-C_(H)2; (xii)V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii) V_(L)-C_(H)2-C_(H)3; and (xiv)V_(L)-C_(L). In any configuration of variable and constant domains,including any of the exemplary configurations listed above, the variableand constant domains may be either directly linked to one another or maybe linked by a full or partial hinge or linker region. A hinge regionmay consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) aminoacids, which result in a flexible or semi-flexible linkage betweenadjacent variable and/or constant domains in a single polypeptidemolecule. Moreover, an antigen-binding domain of a bi-specificantigen-binding molecule may comprise a homo-dimer or hetero-dimer (orother multimer) of any of the variable and constant domainconfigurations listed above in non-covalent association with one anotherand/or with one or more monomeric V_(H) or V_(L) domain (e.g., bydisulfide bond(s)).

The first antigen-binding domain and the second antigen-binding domainmay be directly or indirectly connected to one another to form abi-specific antigen-binding molecule. Alternatively, the firstantigen-binding domain and the second antigen-binding domain may each beconnected to a separate multimerizing domain. The association of onemultimerizing domain with another multimerizing domain facilitates theassociation between the two antigen-binding domains, thereby forming abispecific antigen-binding molecule. As used herein, a “multimerizingdomain” is any macromolecule, protein, polypeptide, peptide, or aminoacid that has the ability to associate with a second multimerizingdomain of the same or similar structure or constitution. For example, amultimerizing domain may be a polypeptide comprising an immunoglobulinC_(H)3 domain. A non-limiting example of a multimerizing component is anFc portion of an immunoglobulin, e.g., an Fc domain of an IgG selectedfrom the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotypewithin each isotype group. In certain embodiments, the multimerizingdomain may be an Fc fragment or an amino acid sequence of 1 to about 200amino acids in length containing at least one cysteine residues. Inother embodiments, the multimerizing domain may be a cysteine residue,or a short cysteine-containing peptide. Other multimerizing domainsinclude peptides or polypeptides comprising or consisting of a leucinezipper, a helix-loop motif, or a coiled-coil motif.

Any bi-specific antibody format or technology may be used to make thebi-specific antigen-binding molecules of the present invention. Forexample, an antibody or fragment thereof having a first antigen bindingspecificity can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmenthaving a second antigen-binding specificity to produce a bi-specificantigen-binding molecule.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H3) domain and a second Ig C_(H3) domain, wherein the first andsecond Ig C_(H3) domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H3) domain binds Protein A and the second Ig C_(H3) domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H3) may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H3) include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bi-specific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mab² bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bi-specific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-Feld1 antibodies or antigen-binding fragments thereof of the presentinvention. The administration of therapeutic compositions in accordancewith the invention will be administered via a suitable route including,but not limited to, intravenously, subcutaneously, intramuscularly,intranasally, with suitable carriers, excipients, and other agents thatare incorporated into formulations to provide improved transfer,delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose of antibody may vary depending upon the age and the size of asubject to be administered, target disease, conditions, route ofadministration, and the like. When the antibody of the present inventionis used for treating the rhinitis or conjunctivitis associated withexposure to a cat, or to cat dander in an individual having asensitivity to Fel d1, or for preventing an anaphylactic response to thecat allergen, or for lessening the severity of the allergic response, itis advantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 30 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. In certain embodiments, the antibody or antigen-bindingfragment thereof of the invention can be administered as an initial doseof at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about5 to about 300 mg, or about 10 to about 200 mg, to about 100 mg, or toabout 50 mg. In certain embodiments, the initial dose may be followed byadministration of a second or a plurality of subsequent doses of theantibody or antigen-binding fragment thereof in an amount that can beapproximately the same or less than that of the initial dose, whereinthe subsequent doses are separated by at least 1 day to 3 days; at leastone week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see, for example, Langer (1990) Science249:1527-1533).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™Autoinjector (Amgen, Thousands Oaks, Calif.), the PENLET™ (Haselmeier,Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA™ Pen (AbbottLabs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

Due to their interaction with Fel d1, the present antibodies are usefulfor treating the primary response following exposure of an individual toa cat, cat dander or to an environment containing the Fel d1 protein, orat least one symptom associated with the allergic response, such asitchy eyes, conjunctivitis, rhinitis, wheezing, breathing difficulties,or for preventing a secondary response to the Fel d1 allergen, includinga more serious anaphylactic response, or for lessening the severity,duration, and/or frequency of symptoms following reexposure to the catallergen. Accordingly, it is envisioned that the antibodies of thepresent invention may be used prophylactically or therapeutically.

In yet a further embodiment of the invention the present antibodies areused for the preparation of a pharmaceutical composition for treatingpatients suffering from a sensitivity to cats, cat dander, cat hair oran extract thereof, and/or the Fel d1 protein. In yet another embodimentof the invention the present antibodies are used for the preparation ofa pharmaceutical composition for reducing the severity of primaryexposure to Fel d1, or for reducing the severity, duration of, and/ornumber of allergic responses to Fel d1. In a further embodiment of theinvention the present antibodies are used as adjunct therapy with anyother agent useful for treating cat allergens, includingcorticosteroids, vaccines, allergen specific immunotherapy (SIT), or anyother palliative therapy known to those skilled in the art.

Combination Therapies

Combination therapies may include an anti-Fel d1 antibody of theinvention and any additional therapeutic agent that may beadvantageously combined with an antibody of the invention, or with abiologically active fragment of an antibody of the invention.

For example, a second therapeutic agent may be employed to aid inreducing the allergic symptoms following exposure to a cat, cat dander,cat hair or an extract thereof, or Fel d1, or being exposed to anenvironment in which a cat resides, such as a corticosteroid. Theantibodies may also be used in conjunction with other therapies, such asa vaccine specific for the Fel d1 allergen. The additionaltherapeutically active component(s) may be administered prior to,concurrent with, or after the administration of the anti-Fel d1 antibodyof the present invention. For purposes of the present disclosure, suchadministration regimens are considered the administration of an anti-Feld1 antibody “in combination with” a second therapeutically activecomponent.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of one or more anti-Fel d1 antibodies (an antibody combination) ora bi-specific antigen-binding molecule may be administered to a subjectover a defined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of an antibody, antibody combination, or a bi-specificantigen-binding molecule of the invention. As used herein, “sequentiallyadministering” means that each dose of an antibody, antibodycombination, or a bi-specific antigen-binding molecule is administeredto the subject at a different point in time, e.g., on different daysseparated by a predetermined interval (e.g., hours, days, weeks ormonths). The present invention includes methods, which comprisesequentially administering to the patient a single initial dose of anantibody, antibody combination, or a bi-specific antigen-bindingmolecule, followed by one or more secondary doses of the antibody, andoptionally followed by one or more tertiary doses of the antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of an antibody, antibodycombination, or a bi-specific antigen-binding molecule of the invention.Thus, the “initial dose” is the dose which is administered at thebeginning of the treatment regimen (also referred to as the “baselinedose”); the “secondary doses” are the doses which are administered afterthe initial dose; and the “tertiary doses” are the doses which areadministered after the secondary doses. The initial, secondary, andtertiary doses may all contain the same amount of an antibody, antibodycombination, or a bi-specific antigen-binding molecule, but generallymay differ from one another in terms of frequency of administration. Incertain embodiments, however, the amount of an antibody, antibodycombination, or a bi-specific antigen-binding molecule contained in theinitial, secondary and/or tertiary doses varies from one another (e.g.,adjusted up or down as appropriate) during the course of treatment. Incertain embodiments, two or more (e.g., 2, 3, 4, or 5) doses areadministered at the beginning of the treatment regimen as “loadingdoses” followed by subsequent doses that are administered on a lessfrequent basis (e.g., “maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21,21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks afterthe immediately preceding dose. The phrase “the immediately precedingdose,” as used herein, means, in a sequence of multiple administrations,the dose of an antibody, antibody combination, or a bi-specificantigen-binding molecule, which is administered to a patient prior tothe administration of the very next dose in the sequence with nointervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an antibody, antibody combination, or a bi-specific antigen-bindingmolecule that specifically binds Fel d1. For example, in certainembodiments, only a single secondary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) secondary doses are administered to the patient. Likewise, incertain embodiments, only a single tertiary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-Fel d1 antibodies of the present invention may also be used todetect and/or measure Fel d1 in a sample, e.g., for diagnostic purposes.It is envisioned that confirmation of an allergic response thought to becaused by Fel d1 may be made by measuring the presence of either Fel d1through use of any one or more of the antibodies of the invention.Exemplary diagnostic assays for Fel d1 may comprise, e.g., contacting asample, obtained from a patient, with an anti-Fel d1 antibody of theinvention, wherein the anti-Fel d1 antibody is labeled with a detectablelabel or reporter molecule or used as a capture ligand to selectivelyisolate Fel d1 protein from patient samples. Alternatively, an unlabeledanti-Fel d1 antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase,horseradish peroxidase, or luciferase. Specific exemplary assays thatcan be used to detect or measure Fel d1 in a sample includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence-activated cell sorting (FACS).

Samples that can be used in Fel d1 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient, which contains detectable quantities of Fel d1 protein, orfragments thereof, under normal or pathological conditions. Generally,levels of Fel d1 in a particular sample obtained from ahealthy/non-allergic patient (e.g., a patient not afflicted with asensitivity associated with the presence of Fel d1) will be measured toinitially establish a baseline, or standard, level of Fel d1. Thisbaseline level of Fel d1 can then be compared against the levels of Feld1 measured in samples obtained from individuals suspected of having asensitivity to Fel d1 in cat dander, or symptoms associated with suchcondition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Fel d1

An immunogen comprising any one of the following can be used to generateantibodies to Fel d1. In certain embodiments, the antibodies of theinvention are obtained from mice immunized with a primary immunogen,such as full length natural Fel d1 (nFel d1), which may be purchasedcommercially (e.g., from Indoor Biotechnologies, # LTN-FD1-1), orisolated from cat hair or dander by multi-step column chromatography(See, for example, Chapman M D, et al. (1988), J. Immunol. 140:812-818),or which may be produced recombinantly (See GenBank accession numbersP30438, or NP_001041618.1 for the full length amino acid sequence ofchain 1 of Fel d1 (also referred to as chain A or FELD1 A; also see SEQID NO: 392) and GenBank accession number P30440, or NP_001041619.1 forthe full length amino acid sequence of chain 2 of Fel d1 (also referredto as chain B or FELD B; also see SEQ ID NO: 393), or fragments ofeither chain 1 or chain 2, or fragments from both chain 1 and chain 2 ofthe Fel d1 protein, followed by immunization with a secondary immunogen,or with an immunogenically active fragment of the natural protein.Animals may be immunized with either chain 1 protein alone or chain 2protein alone, or with both chain 1 and chain 2 proteins, administeredsequentially, or concurrently. Various constructs may be prepared usingportions of chain 1 and chain 2 along with various linking or spacerstrategies known to those skilled in the art. These constructs may beused alone, or in various combinations to elicit antibody responses invivo. For example, recombinant Fel d1 constructs, such as thoseexemplified in SEQ ID NOs: 385, 394, 395, 396 or 397, or fragmentsthereof, may be used as immunogens.

In certain embodiments, the antibodies of the invention are obtainedfrom mice immunized with a primary immunogen, such as a biologicallyactive and/or immunogenic fragment of natural Fel d1, or DNA encodingthe active fragment thereof. The fragment may be derived from theN-terminal or C-terminal domain of either chain 1 and/or chain 2 of Feld1.

In certain embodiments, the recombinantly produced Fel d1 immunogen maybe made by direct fusion of the two chains of Fel d1, as described inKaiser et. al., to produce a fusion product that has a similar refoldingpattern to that of natural Fel d1 (Kaiser, L. et al., (2003), J. Biol.Chem. 278(39):37730-37735). In certain embodiments, the immunogen may bea fusion protein such as that shown in the constructs of SEQ ID NOs:385, 394, 395, 396 or 397, followed by immunization with a secondaryimmunogen, or with an immunogenically active fragment of the natural orrecombinantly produced Fel d1.

In certain embodiments, the recombinant Fel d1 protein constructs usedin the studies described herein are comprised of either i) Fel d1 Bchain (chain 2) and Fel d1 A chain (chain 1) linked as a continuous,in-line fusion (with Fel d1 B chain at the N-terminus) or ii) acontinuous, in-line fusion with Fel d1 A chain at the N-terminusfollowed by a flexible linker [(Gly4Ser)₃] followed by Fel d1 B. Theseconstructs may also include a C-terminal tag (myc-myc-His6 or mouseIgG2a Fc region), as indicated below. The proteins were expressed inChinese hamster ovary (CHO) cells. An exogenous signal sequence used topromote expression in CHO cells is not included in the sequencelistings.

In certain embodiments, the immunogen may be a fusion protein comprisingany one or more of the following: i) amino acid residues 18-109 of chain2 of Fel d1 (See GenBank accession number P30440 and also SEQ ID NO:393) fused via the C terminus directly with the N terminus of amino acidresidues 23-92 of chain 1 of Fel d1 (See GenBank accession number P30438and also SEQ ID NO: 392); ii) amino acid residues 23-92 of chain 1 ofFel d1 (See GenBank accession number P30438 and also SEQ ID NO: 392)fused via the C terminus to the N terminus of amino acid residues 18-109of chain 2 of Fel d1 (See GenBank accession number P30440 and also SEQID NO: 393); iii) amino acid residues 18-109 of chain 2 of Fel d1 (SeeGenBank accession number NP_001041619.1) fused via the C terminusdirectly with the N terminus of amino acid residues 19-88 of chain 1 ofFel d1 (See GenBank accession number NP_001041618), such as theconstruct shown in SEQ ID NO: 394 or 396; iv) amino acid residues 19-88of chain 1 of Fel d1 (See GenBank accession number NP_001041618.1) fusedvia the C terminus to the N terminus of amino acid residues 18-109 ofchain 2 of Fel d1 (See GenBank accession number NP_001041619.1). Seealso SEQ ID NO: 395). In certain embodiments, the fusion protein mayhave a tag at the C terminal end of the construct, such as amyc-myc-hexahistidine tag (See SEQ ID NOs: 385, 396 or 397 for suchconstructs.). In related embodiments, the fusion protein may have amouse Fc coupled at the C terminal end of the construct (See SEQ ID NOs:394 or 395 for such constructs.). In certain embodiments, chains 1 and 2are coupled via a linker known to those skilled in the art, e.g. (G₄S)₃(See SEQ ID NOs: 395 and 397 for such a construct.).

In certain embodiments, antibodies that bind specifically to Fel d1 maybe prepared using fragments of the above-noted regions, or peptides thatextend beyond the designated regions by about 5 to about 20 amino acidresidues from either, or both, the N or C terminal ends of the regionsdescribed herein. In certain embodiments, any combination of theabove-noted regions or fragments thereof may be used in the preparationof Fel d1 specific antibodies. In certain embodiments, any one or moreof the above-noted regions of Fel d1, or fragments thereof may be usedfor preparing monospecific, bispecific, or multispecific antibodies.

The full length proteins, or fragments thereof, that were used asimmunogens, as noted above, were administered directly, with an adjuvantto stimulate the immune response, to a VELOCIMMUNE® mouse comprising DNAencoding human Immunoglobulin heavy and kappa light chain variableregions. The antibody immune response was monitored by a Fel d1-specificimmunoassay. When a desired immune response was achieved splenocyteswere harvested and fused with mouse myeloma cells to preserve theirviability and form hybridoma cell lines. The hybridoma cell lines werescreened and selected to identify cell lines that produce Fel d1specific antibodies. Using this technique, and the various immunogensdescribed above, several anti-Fel d1, chimeric antibodies (i.e.,antibodies possessing human variable domains and mouse constant domains)were obtained; certain exemplary antibodies generated in this mannerwere designated as H1M1230N, H1M1234N, H1M1241N, H2M1233N, H2M1236N,H2M1237N, and H2M1242N.

Anti-Fel d1 antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in U.S.2007/0280945A1, herein specifically incorporated by reference in itsentirety. Using this method, several fully human anti-Fel d1 antibodies(i.e., antibodies possessing human variable domains and human constantdomains) were obtained; exemplary antibodies generated in this mannerwere designated as follows: H4H2574P, H4H2590S, H4H2592B, H4H2594S,H4H2597P, H4H2606B, H4H2607B, H4H2608B, H4H2636P, H4H2645P, H4H2793P,H4H2797P and H4H2864P.

The biological properties of the exemplary antibodies generated inaccordance with the methods of this Example are described in detail inthe Examples set forth below.

Example 2 Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs of selected antibodies specific for Fel d1 and theircorresponding antibody identifiers. Antibodies are typically referred toherein according to the following nomenclature: Fc prefix (e.g. “H4H”,“H1M, “H2M”), followed by a numerical identifier (e.g. “1232” as shownin Table 1), followed by a “P” or “N” suffix. Thus, according to thisnomenclature, an antibody may be referred to as, e.g. “H1M1232N”. TheH4H, H1M, and H2M prefixes on the antibody designations used hereinindicate the particular Fc region of the antibody. For example, an “H2M”antibody has a mouse IgG2 Fc, whereas an “H4H” antibody has a human IgG4Fc. As will be appreciated by a person of ordinary skill in the art, anH1M or H2M antibody can be converted to an H4H antibody, and vice versa,but in any event, the variable domains (including the CDRs), which areindicated by the numerical identifiers shown in Table 1, will remain thesame. Antibodies having the same numerical antibody designation, butdiffering by a letter suffix of N, B, S or P refer to antibodies havingheavy and light chains with identical CDR sequences but with sequencevariations in regions that fall outside of the CDR sequences (i.e., inthe framework regions). Thus, N, B, S and P variants of a particularantibody have identical CDR sequences within their heavy and light chainvariable regions but differ from one another within their frameworkregions.

TABLE 1 Antibody AMINO ACID SEQ ID NOs: Designation HCVR HCDR1 HCDR2HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1M1230N 2 4 6 8 10 12 14 16 H4H1232N 18 2022 24 26 28 30 32 H1M1234N 34 36 38 40 42 44 46 48 H1M1241N 50 52 54 5658 60 62 64 H4H1300N 66 68 70 72 74 76 78 80 H2M1233N 82 84 86 88 90 9294 96 H2M1236N 98 100 102 104 106 108 110 112 H2M1237N 114 116 118 120122 124 126 128 H4H1238N 130 132 134 136 138 140 142 144 H2M1242N 146148 150 152 154 156 158 160 H4H1616N 162 164 166 168 170 172 174 176H4H2574P 178 180 182 184 186 188 190 192 H4H2590S 194 196 198 200 202204 206 208 H4H2592B 210 212 214 216 218 220 222 224 H4H2594S 226 228230 232 234 236 238 240 H4H2597P 242 244 246 248 250 252 254 256H4H2606B 258 260 262 264 266 268 270 272 H4H2607B 274 276 278 280 282284 286 288 H4H2608B 290 292 294 296 298 300 302 304 H4H2636P 306 308310 312 314 316 318 320 H4H2645P 322 324 326 328 330 332 334 336H4H2793P 338 340 342 344 346 348 350 352 H4H2797P 354 356 358 360 362364 366 368 H4H2864P 370 372 374 376 378 380 382 384 H4H2574B 428 430432 434 436 438 440 442 H4H2597B 444 446 448 450 452 454 456 458H4H2636B 460 462 464 466 468 470 472 474 H4H2645B 476 478 480 482 484486 488 490

Example 3 Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage (V_(H), D, J_(H), V_(K), or J_(K)) was identifiedfor each Heavy Chain Variable Region (HCVR) and Light Chain VariableRegion (LCVR). Table 2 sets forth the gene usage for selected antibodiesin accordance with the invention.

TABLE 2 Antibody Antibody Identifier HCVR LCVR PID HCVR/LCVR V_(H) DJ_(H) V_(K) J_(K) H1M1230N  2/10 3-7 6-13 6 1-12 5 H4H1232N 18/26 3-212-15 6 1-27 2 H1M1234N 34/42 6-1 1-7 4 4-1 4 H1M1241N 50/58 3-21 2-2 61-17 4 H4H1300N 66/74 1-2 5-12 4 4-1 2 H2M1233N 82/90 3-33 6-19 4 1-5 1H2M1236N  98/105 4-59 1-7 4 1-33 2 H2M1237N 114/122 3-33 6-19 4 1-5 1H4H1238N 130/138 4-59 1-7 4 1-33 2 H2M1242N 146/154 3-21 5-12 4 1-5 1H4H1616N 162/170 3-23 6-13 4 1-33 3 H4H2574P 178/186 4-39 6-19 3 3-20 2H4H2590S 194/202 3-11 6-6 4 6-21 1 H4H2592B 210/218 3-11 1-26 4 6-21 1H4H2594S 226/234 3-11 6-6 4 1-16 4 H4H2597P 242/250 3-11 6-6 4 6-21 1H4H2606B 258/266 3-11 3-9 4 6-21 1 H4H2607B 274/282 3-11 1-26 4 1-17 2H4H2608B 290/298 3-11 1-26 4 6-21 1 H4H2636P 306/314 3-23 1-1 4 1-5 4H4H2645P 322/330 3-23 ND 1 1-16 3 H4H2793P 338/346 3-7 3-16 4 1-12 1H4H2797P 354/362 3-33 5-12 3 1-16 3 H4H2864P 370/378 3-23 1-7 4 1-9 3

Example 4 Antibody Binding to Fel d1 as Determined by Surface PlasmonResonance

Binding associative and dissociative rate constants (k_(a) and k_(d),respectively) and calculated equilibrium dissociation constants anddissociative half-lives (K_(D) and t_(1/2), respectively) for antigenbinding to anti-Fel d1 monoclonal antibodies were determined using areal-time surface plasmon resonance biosensor (Biacore T200 or Biacore2000) assay. The Biacore sensor surface was derivatized with eitherpolyclonal rabbit anti-mouse antibody (GE Healthcare, # BR-1008-38) orwith monoclonal mouse anti-human Fc antibody (GE Healthcare, #BR-1008-39) to capture anti-Fel d1 antibodies, expressed with mouse Fc(antibody ID prefix H1M, H2M, H2aM, H2bM) or human IgG4 Fc (antibody IDprefix H4H), respectively. For kinetic fits, at least two differentconcentrations (ranging from 390 pM to 67 nM) of natural Fel d1 (IndoorBiotech, # NA-FD1-2) or a recombinant version of the protein, Fel d1(B-A)-mmH (SEQ ID NO: 396) were injected over the anti-Fel d1 monoclonalantibody-captured surface at 25° C. at a flow rate of 50 μl/min inrunning buffer (10 mM HEPES, 150 mM NaCl, 0.05% P20, 3 mM MgCl₂, 3 mMCaCl₂). Fel d1 (B-A)-mmH was expressed in Chinese hamster ovary (CHO)cells and is comprised of amino acids 18-109 of Fel d1 B (accession#P30440) fused in-line with amino acids 23-92 of Fel d1 A (accession#P30438) with a C-terminal myc-myc-hexahistidine tag. Antibody-antigenassociation was monitored for 3 to 5 minutes, and the dissociation ofantigen from the captured monoclonal antibody (in running buffer aloneat 25° C.) was monitored for 10 or 15 minutes. Kinetic association(k_(a)) and dissociation (k_(d)) rate constants were determined byprocessing and fitting the data to a 1:1 binding model using Scrubber2.0 curve fitting software. Binding dissociation equilibrium constants(K_(D)) and dissociative half-lives (t₁₂) were calculated from thekinetic rate constants as: K_(D)=k_(d)/k_(a) and t_(1/2)=In(2)/k_(d).Binding parameters for different anti-Fel d1 monoclonal antibodies aretabulated in Table 3 and Table 4. Table 3 shows the Biacore affinitiesat 25° C. for natural Fel d1 binding to captured anti-Fel d1 monoclonalantibodies and Table 4 shows the Biacore affinities at 25° C. forrecombinant Fel d1 binding to captured anti-Fel d1 monoclonalantibodies.

As shown in Table 3, 10 of the 25 antibodies tested exhibited K_(D)values below 1 nM for binding to natural Fel d1, ranging from 207 pM to982 pM. As shown in Table 4, 17 of the 25 antibodies tested exhibitedK_(D) values below 1 nM for binding to recombinant Fel d1, ranging from144 pM to 924 pM. Two of the antibodies, H4H2574B and H4H2793P, bound torecombinant, but not natural Fel d1 under these experimental conditions

TABLE 3 mAb Captured k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) t_(1/2) (min)H4H1232N 1.60E+06 3.31E−04 2.07E−10 35 H4H1238N 3.83E+05 1.67E−034.37E−09 7 H4H1300N* 4.40E+04 2.11E−01 4.80E−06 0.05 H4H1616N 2.41E+051.24E−03 5.14E−09 9 H1M1230N 2.58E+05 8.69E−04 3.37E−09 13 H1M1234N3.71E+05 6.79E−03 1.83E−08 2 H2M1233N 2.53E+05 3.53E−04 1.40E−09 33H2M1236N 3.12E+05 1.42E−03 4.55E−09 8 H2M1237N 2.81E+05 2.76E−049.82E−10 42 H1M1241N 1.82E+05 6.62E−04 3.63E−09 17 H2M1242N 1.92E+056.04E−04 3.14E−09 19 H4H2574B NB NB NB NB H4H2590S 1.23E+06 8.02E−046.55E−10 14 H4H2592B 1.14E+06 7.28E−04 6.41E−10 16 H4H2594S 1.10E+069.65E−04 8.78E−10 12 H4H2597B 2.31E+06 1.50E−03 6.50E−10 8 H4H2606B9.24E+05 7.07E−04 7.65E−10 16 H4H2607B 2.97E+06 9.10E−04 3.07E−10 13H4H2608B 5.16E+05 1.06E−03 2.05E−09 11 H4H2636B 2.24E+05 3.95E−041.77E−09 29 H4H2793P NB NB NB NB H4H2797P 2.02E+05 7.13E−03 3.54E−08 2H4H2864P 1.68E+06 1.35E−03 8.01E−10 9 H4H2645P 5.69E+05 2.61E−044.59E−10 44 H4H2636P 4.31E+05 4.48E−04 1.04E−09 26 *Because of the lowerobserved binding affinity, higher injected concentrations of natural Feld1 (67 nM, 200 nM, and 600 nM) were used for this sample.

TABLE 4 mAb Captured k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) t_(1/2) (min)H4H1232N 1.79E+06 2.58E−04 1.44E−10 45 H4H1238N 7.35E+05 9.74E−041.33E−09 12 H4H1300N* 1.68E+05 2.28E−01 1.36E−06 0.05 H4H1616N 2.29E+051.88E−03 8.21E−09 6 H1M1230N 3.88E+05 5.10E−04 1.31E−09 23 H1M1234N2.54E+05 1.42E−03 5.58E−09 8 H2M1233N 3.05E+05 1.66E−04 5.44E−10 70H2M1236N 4.15E+05 2.32E−04 5.58E−10 50 H2M1237N 3.59E+05 1.65E−044.58E−10 70 H1M1241N 3.37E+05 1.12E−04 3.31E−10 104 H2M1242N 2.72E+051.22E−04 4.49E−10 94 H4H2574B 1.25E+05 3.73E−04 2.98E−09 31 H4H2590S1.31E+06 4.16E−04 3.18E−10 28 H4H2592B 1.55E+06 5.56E−04 3.58E−10 21H4H2594S 1.30E+06 5.21E−04 4.02E−10 22 H4H2597B 1.12E+06 5.58E−045.01E−10 21 H4H2606B 1.26E+06 4.86E−04 3.88E−10 24 H4H2607B 1.55E+065.63E−04 3.64E−10 21 H4H2608B 9.70E+05 5.89E−04 6.07E−10 20 H4H2636B2.47E+05 2.28E−04 9.24E−10 51 H4H2793P 1.52E+05 1.95E−04 1.28E−09 59H4H2797P 4.37E+05 2.05E−03 4.69E−09 6 H4H2864P 5.37E+05 3.09E−045.76E−10 37 H4H2645P 4.87E+05 1.79E−04 3.68E−10 65 H4H2636P 2.57E+052.35E−04 9.12E−10 49 *Because of the lower observed binding affinity,higher injected concentrations of recombinant Fel d1 (67 nM, 200 nM, and600 nM) were used for this sample

Example 5 Cross Competition of Anti-Fel d1 Antibodies for Binding toNatural (n) Fel d1

A binding experiment was performed using an Octet Red biosensor system(Fortebio Inc.) to determine cross-competition for a panel of 8 anti-Feld1 antibodies binding to natural Fel d1 (nFel d1; IndoorBiotechnologies, #NA-FD1-2). The experiment was performed at 25° C. inHBST buffer (0.01 M HEPES pH7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/vSurfactant P20) containing 0.1 mg/mL BSA. A washing step with the HBSTbuffer was performed between each binding step, and plates were agitatedduring the binding and washing steps using an orbital plate shaker at1000 rpm. A first anti-Fel d1 antibody (mAb-1) was captured for 2minutes onto the anti-hFc biosensor surface from stock solutions ofantibody at 10 ug/mL (final capture levels ˜1.5 nm response units). Thecoated sensor tips were then blocked for 5 minutes with a 100 ug/mLsolution of an irrelevant antibody. Sensor tips were then submerged intowells containing 500 nM of nFel d1 for 5 minutes, and then into wellscontaining 50 ug/mL solutions of a second anti-Fel d1 antibody (mAb-2).The mAb-2 solutions were supplemented with 100 ug/mL of an irrelevantantibody to minimize non-specific binding. The binding responses formAb-2 binding to nFel d1 pre-complexed with mAb-1 were measured for the8×8 antibody matrix (Table 5). Each binding value for mAb-2 binding to adifferent mAb-1/Fel d1 capture surface (down a column in Table 5) wassubtracted by the mAb-1/Fel d1/mAb-2 self-competition value (wheremAb-1=mAb-2; across the diagonal in Table 5). Values below 0.10 nmindicate cross-competition of mAb-1 and mAb-2 to a common binding siteon Fel d1.

Four antibodies, H4H2636P, H4H1616N, H4H2645P, and H4H2864P,bi-directionally compete with each other for binding to nFel d1, but donot compete with any of the other anti-Fel d1 antibodies. Twoantibodies, H4H1232N and H4H2597P, bi-directionally compete with eachother for binding to nFel d1. Both H4H1232N and H4H2597Puni-directionally compete with H4H1300N. Bi-directional competition withH4H1300N could not be determined because H4H1300N did not pre-complexwith nFel d1. H4H1238N did not compete with any of the anti-Fel d1antibodies for binding to nFel d1.

TABLE 5 Amount of Amount 500 nM of mAb-1 nFel d1 Response of mAb-2Binding to nFel d1 mAb Captured +/− Std Bound +/− Std dev pre-complexedwith mAb-1 (nm) Captured dev (nm) (nm) H4H2636P H4H1616N H4H2645PH4H2636P 1.37 ± 0.07 0.09 ± 0.01 0.00 0.01 0.00 H4H1616N 1.21 ± 0.080.09 ± 0.01 −0.01 0.00 −0.01 H4H2645P 1.31 ± 0.07 0.10 ± 0.01 0.00 0.010.00 H4H2864P 1.41 ± 0.09 0.08 ± 0.01 −0.01 0.01 −0.01 H4H2597P 1.26 ±0.08 0.06 ± 0.01 0.55 0.40 0.55 H4H1232N 1.51 ± 0.09 0.08 ± 0.02 0.760.54 0.75 H4H1238N 1.28 ± 0.08 0.08 ± 0.01 0.63 0.43 0.62 H4H1300N 1.29± 0.09 −0.02 ± 0.01  0.06 0.06 0.05 Response of mAb-2 Binding to nFel d1pre-complexed with mAb-1 (nm) mAb Captured H4H2864P H4H2597P H4H1232NH4H1238N H4H1300N H4H2636P 0.00 0.27 0.29 0.28 0.38 H4H1616N −0.01 0.180.18 0.21 0.20 H4H2645P −0.01 0.29 0.31 0.31 0.26 H4H2864P 0.00 0.270.30 0.30 0.33 H4H2597P 0.54 0.00 −0.03 0.56 −0.03 H4H1232N 0.71 0.000.00 0.73 −0.02 H4H1238N 0.62 0.65 0.76 0.00 0.60 H4H1300N 0.06 0.030.02 0.07 0.00

Example 6 Effect of Anti-Fel d1 Antibodies in a Passive CutaneousAnaphylaxis (PCA) In Vivo Model

The passive cutaneous anaphylaxis (PCA) in vivo model was used to assessin vivo mast cell degranulation. The model involves intradermalinjection of an allergen-specific antiserum into a local area on theskin followed by intravenous injection of an antigen along with a dye.The allergic reaction causes capillary dilatation and increased vascularpermeability at the site of sensitization, resulting in preferentialaccumulation of dye at this site. The dye can be extracted from thetissue and quantitated spectrophotometrically. Dye extravasation intotissue sensitized with test antiserum is compared to extravasation intotissue sensitized with a non-relevant antiserum.

Antisera were generated by immunizing Balb/c mice with 5 μg natural Feld1 protein purified from cat hair extract (Indoor Biotechnologies, #LTN-FD1-1), 5 μg of crude peanut allergen extract (Greer Laboratories, #XPF171D3A25), or 1250 of Bioequivalent allergy units (BAU) ofstandardized cat hair extract (Greer Laboratories, #GTE3A01) in asolution of 1 mg/ml of alum (Pierce, #77161) in 1× phosphate bufferedsaline. Two weeks later (day 14) sensitized mice were boosted with dosesof allergen identical to those used for the initial immunization. Twoweeks after the boost (day 28), mice were sacrificed and serum wascollected. Total IgE concentration in the isolated antisera wasdetermined by ELISA. The final concentration of antiserum was diluted to2400 ng/mL IgE in 1× phosphate buffered saline.

To determine the effect of anti-Fel d1 antibodies on mast celldegranulation in the PCA model, prior to ear sensitization withantiserum generated as described above, groups of Balb/c mice were firstinjected subcutaneously with either a human IgG4 isotype controlantibody, an anti-Fel d1 antibody, or a combination of anti-Fel d1antibodies at doses of 5 mg/kg (total antibody dose, 2.5 mg/kg of eachantibody) for single point experiments unless otherwise indicated or atconcentrations ranging from 0.06 mg/kg to 2 mg/kg for dose-rangingexperiments. Three days after pre-treatment with antibodies, one groupof mice (“natural Fel d1 group”) was sensitized by intradermal injectionwith 10 μl of natural Fel d1-derived antiserum or 10 μl ofpeanut-derived antiserum (negative control) into the right and leftears, respectively, of each mouse. A second group of mice (“cat extractgroup”) was sensitized with 20 μL of cat hair extract-derived antiserumor 20 μL of peanut-derived antiserum (negative control) into the rightand left ears, respectively, of each mouse. Twenty-four hours aftersensitization, mice in the natural Fel d1 group were challenged byintravenous injection (100 μL per mouse) of a solution of 0.25 μg/mLnatural Fel d1 (Indoor Biotechnologies, # LTN-FD1-1) dissolved in 1×phosphate buffered saline containing 0.5% (w/v) Evan's blue dye (Sigma,# E2129). Similarly, 24 hours after sensitization, mice in the catextract group were challenged with 250BAU of standardized cat hairextract [standardized cat hair extract (Greer Laboratories, #GTE3A01)]dissolved in 1× phosphate buffered saline containing 0.5% (w/v) Evan'sblue dye (Sigma, # E2129). One hour after antigen challenge, mice weresacrificed, ears were excised and placed in 1 mL formamide and incubatedfor 3 days at 56° C. to extract the Evan's blue dye from the tissue. Eartissue was then removed from the formamide, blotted to remove excessliquid and weighed. Two hundred microliter aliquots of each formamideextract were transferred to 96 well plates in duplicate. Absorbance ofthe resulting supernatants was measured at 620 nm. The OD was convertedto Evan's blue dye concentration using a standard curve. The averageconcentration of Evan's blue dye extravasated into the tissue of theantisera-sensitized ear (normalized by ear tissue weight) was calculatedfor the group treated with the isotype control antibody and defined asF(isotype,avg). The reduction in Evan's blue dye extravasation resultingfrom antibody pre-treatment was calculated per mouse by subtracting theamount of Evan's blue dye for the antibody-treated group's Fel d1 orextract sensitized ear, defined as F(mAb,i), from F(isotype,avg). Thisnumber was then divided by the difference between F(isotype,avg) and thedye amount for the antibody-treated group's peanut sensitized ear[P(mAb,i)] and multiplied by 100 to give the overall percent reductionin dye extravasation for each mouse (% Reduction).% Reduction (permouse)=100*[F(isotype,avg)−F(mAb,i)]/[F(isotype,avg)−P(mAb,i)]

The average percent reduction in dye leakage was then calculated foreach antibody group. Results, expressed as (mean±SD) of percent Evan'sblue reduction are shown in Table 6 and Table 7 for the natural Fel d1group and in Table 8 for the cat hair extract group.

As shown in Table 6, seven groups of mice from the natural Fel d1 group,when treated with specific combinations of anti-Fel d1 antibodies atfixed concentrations, exhibited reductions in dye extravasations rangingfrom 79% to 103% compared to mice receiving control antibody. Micetreated with H4H2590S/H4H1238N, H4H2590S/H4H2574P, or H4H1232N/H4H1616Npairwise antibody combinations exhibited less than 3% reduction in dyeextravasation compared to mice receiving control antibody, demonstratingthat not all anti-Fel d1 antibodies tested in this model wereefficacious.

In addition, dose-ranging experiments were performed with mice from thenatural Fel d1 group, as shown in Table 7. Single antibodies were not aseffective at reducing dye extravasation as the anti-Fel d1 antibodycombinations at the tested doses.

A specific pair of anti-Fel d1 antibodies (H4H2636P and H4H1232N) atmultiple dose levels, as well as each of these anti-Fel d1 antibodiesalone at a single (highest) dose level, was further tested in the PCAmodel using mice that were sensitized and challenged with cat hairextract as shown in Table 8. At 2 mg/kg, these single anti-Fel d1antibodies alone were not as efficacious at reducing dye extravasationas a combination of the two antibodies. The combination of H4H2636P andH4H1232N at both 2 mg/kg and 1 mg/kg reduced dye extravasation by morethan 90% as compared with the isotype control in the PCA model using cathair extract as the antigen.

All reductions that were statistically significant (p<0.05) compared toisotype control as determined by two-way ANOVA with Bonferroni'spost-test are noted with an asterisk (*). The number of mice used pergroup (n) is noted within parentheses in the tables.

TABLE 6 % Reduction in Dye Antibody Extravasation H4H1232N + H4H1238N*(n = 5) 87 ± 8  H4H1232N + H4H2645B* (n = 5) 87 ± 29 H4H1232N +H4H2636B* (n = 5) 89 ± 23 H4H1232N + H4H2864P* (n = 5) 79 ± 27H4H1232N + H4H1238N + H4H1300N + 103 ± 16  H4H1616N* (n = 5) H4H1232N +H4H1616N (n = 5)§  3 ± 92 H4H2590S + H4H1238N (n = 5)  0 ± 74 H4H2597P +H4H2636P*^(,)** (n = 5) 89 ± 4  H4H2597P + H4H2645P*^(,)** (n = 5) 85 ±36 H4H2590S + H4H2574P (n = 5)  0 ± 129 § 10 mg/kg total antibodyconcentration; **0.5 mg/kg total antibody concentration

TABLE 7 Percent Reduction in Dye Extravasation Antibodies used 1 mg/kg0.5 mg/kg 0.25 mg/kg 0.125 mg/kg 0.06 mg/kg Study 1 H4H1232N + H4H2636P 84 ± 16*  53 ± 41*  53 ± 40* 19 ± 32 (n = 15) (n = 15) (n = 15) (n =15) H4H1232N 24 ± 61 (n = 15) H4H2636P  0 ± 44 (n = 15) Study 2H4H1232N + H4H2645P 66 ± 29 49 ± 37 22 ± 36 0.26 ± 0.28 (n = 10) (n =10) (n = 10) (n = 10) H4H1232N 6 ± 6 (n = 10) H4H2645P 14 ± 14 (n = 10)Study 3 H4H1232N + H4H2864P  93 ± 10*  50 ± 33*  49 ± 42* 11 ± 28 (n =10) (n = 9) (n = 10) (n = 10) H4H1232N  0 ± 45 (n = 10) H4H2864P  0 ± 35(n = 10) Study 4 H4H1232N + H4H1238N 46 ± 46  60 ± 19*  48 ± 53*  0 ± 47(n = 10) (n = 10) (n = 10) (n = 10) H4H1232N 21 ± 57 (n = 10) H4H1238N35 ± 36 (n = 10) Study 5 H4H2597P + H4H2636P 90 ± 8*  81 ± 16* 43 ± 2114 ± 32 (n = 5) (n = 5) (n = 5) H4H2597P  0 ± 49 (n = 5) H4H2636P 28 ±51 (n = 5) Study 6 H4H2597P + H4H2645P  64 ± 41* 27 ± 25 18 ± 39  0 ± 16(n = 10) (n = 5) (n = 5) (n = 5) H4H2597P  7 ± 31 (n = 5) H4H2645P  0 ±26 (n = 5)

TABLE 8 Percent Reduction in Dye Extravasation Antibodies used 2 mg/kg 1mg/kg 0.5 mg/kg 0.25 mg/kg H4H1232N + H4H2636P 93 ± 4* 97 ± 2* 85 ± 13*40 ± 51 (n = 5) (n = 5) (n = 5) (n = 5) H4H1232N 66 ± 9* (n = 5)H4H2636P 40 ± 55 (n = 5)

Example 7 Effect of Anti-Fel d1 Antibodies in a Lung Inflammation InVivo Model

The lung inflammation in vivo mouse model is used to assess allergeninduced lung inflammation and mucus accumulation that could beassociated with asthma or rhinoconjuctivitis. The model involvesrepeated intranasal administration of an allergen into previouslyallergen-sensitized mice. The allergen-associated inflammation can causeincreases in lung mucus accumulation, eosinophil migration into thelung, serum total IgE, and allergen specific IgG1 levels.

Balb/c mice were intraperitoneally immunized with 1 ug of natural Fel d1 protein purified from cat hair extract (Indoor Biotechnologies,#LTN-FD1-1) in a solution of 1 mg/mL of alum (Pierce, #77161) in 1×phosphate buffered saline. Seven days later, sensitized mice wereboosted intraperitoneally with 1 ug of natural Fel d 1 in a solution of1 mg/mL alum in 1× phosphate buffered saline. On days 17, 21, and 25,groups of mice (n=5) were injected subcutaneously with a human IgG4isotype control antibody or a 1:1 combination of anti-Fel d 1antibodies, H4H1232N and H4H2636P, at 20 mg/kg (total antibody dose). Ondays 20, 24, and 28, mice were intranasally challenged with 0.05 ug ofnatural Fel d 1 diluted in 20 uL of 1× phosphate buffered saline.Control mice were challenged with 20 uL of 1× phosphate buffered salineon the same days. On day 32, all mice were sacrificed and their lungswere harvested. Experimental dosing and treatment protocol for groups ofmice are shown in Table 9.

To determine circulating total IgE and Fel d 1 specific IgG1 in theserum of the mice, serum samples were collected for each mouse viaterminal cardiac puncture using a 27G1/2 1 mL TB syringe (BectonDickinson, #309306) with a needle attached. Blood samples were placedinto BD Microtainer® serum separator tubes (Becton Dickinson, #365956),centrifuged, and then the serum was transferred to a fresh tube forstorage until analysis.

To determine the total IgE concentration in the serum samples for eachmouse, a sandwich ELISA OPTEIA kit (BD Biosciences, #555248) was usedaccording to the manufacturer's instructions. Serum samples were dilutedand incubated with anti-IgE capture antibody coated on 96-well plates.Total IgE was detected by biotinylated anti-mouse IgE secondaryantibody. Purified horseradish peroxidase (HRP)-labeled mouse IgE wasused as a standard. The chromagen 3,3′,5,5′-tetramethylbenzidine (TMB)(BD OPTEIA substrate reagent set, BD, #555214) was used to detect HRPactivity. A stop solution of 1M sulfuric acid was then added, andabsorbance at 450 nm was measured on a Molecular Devices SpectraMax M5plate reader. Data analysis was performed using Prism™ software. Themean amounts of circulating IgE levels in serum for each experimentalgroup are expressed as ng/mL (±SEM) as shown in Table 10. Micechallenged with Fel d 1 intranasally when treated with the combinationof anti-Fel d 1 antibodies exhibited a significant decrease in theamount of circulating IgE [6683 (±1394) ng/mL] compared to micereceiving isotype control antibody [14080 (±1505) ng/mL].

To determine the Fel d 1 specific IgG1 levels in the serum samples fromeach mouse, an ELISA was utilized. Fel d 1 coated plates were incubatedwith serially diluted mouse serum samples, followed by incubation withanti-mouse IgG1-HRP conjugated antibody (BD Biosciences, #559626). Allsamples were developed with a TMB solution and analyzed as describedabove. Relative levels of circulating IgG1 in serum were represented astiter units (titer units were calculated by multiplying the measured ODby a dilution factor required to achieve OD450 that was greater than twotimes background). The mean circulating Fel d 1-specific IgG1 levels inserum for each experimental group are expressed as titer×10³ (±SEM) asshown in Table 11. Mice challenged with Fel d 1 intranasally whentreated with the combination of anti-Fel d 1 antibodies exhibited asignificant decrease in the amount of Fel d 1-specific IgG1 levels inserum [titer of 105.3 (±31.33)×10³] when compared to mice receivingisotype control antibody [titer of 526.1 (±144.0)×10³].

Lung Harvest for Cell Infiltrate Analysis:

After exsanguination, the right lung from each mouse was removed andplaced into a small petri dish containing Dulbecco's Modified EagleMedium (DMEM) (Irvine Scientific, #9033) and chopped into cubes thatwere approximately 2 to 3 mm in size. The cubes were then transferred toa tube containing a solution of 20 μg/mL DNAse (Roche, #10104159001) and0.7 U/mL Liberase TH (Roche, #05401151001) diluted in Hank's BalancedSalt Solution (HBSS) (Gibco, #14025) and placed into a 37° C. water bathfor 20 minutes with vortexing every 5 minutes. This reaction was thenstopped by adding ethylenediaminetetraacetic acid (EDTA) (Gibco, #15575)at a final concentration of 10 mM. Each lung was mashed, filteredthrough a 70 μm filter, centrifuged, and then lung pellet wasresuspended in 4 mL of ACK lysing buffer (Gibco, #10492) to remove redblood cells. After a 3 minute room temperature incubation, DMEM wasadded to deactivate the ACK buffer. The cell suspensions werecentrifuged, and the cell pellets were then resuspended into 10 mL ofMACS buffer solution [a mixture of Militenyi auto MACS Rinsing Solution(Militenyi Biotec, #130-091-222) and MACS BSA (Militenyi Biotec,#130-091-376)]. The resuspended samples were filtered through a 70 μmfilter and 1×10⁶ cells were plated into a 96-well V-bottom plate. Cellswere then centrifuged and the pellets were resuspended in purified ratanti-mouse CD16/CD32 Fc Block, (BD Biosciences Clone: 2.4G2, #553142)diluted in MACS Buffer for 15 minutes at 40° C. The cells were washedtwice and were then incubated in the appropriate antibody mixture(described in Table 12) diluted in MACS buffer for 30 minutes at 4° C.protected from light. After antibody incubation, the cells were washedtwice in MACS buffer and resuspended in BD cytofix (BD Biosciences,#554655) for 15 minutes at 4° C. while being protected from light. Thecells were washed, resuspended in MACS buffer and were then transferredto BD FACS tubes (BD Biosciences, #352235) for analysis of eosinophilsby flow cytometry. Eosinophils were defined as cells that were CD45⁺,GR1⁻, CD11c^(lo), SiglecF^(hi). Data are expressed as frequency ofeosinophils in CD45⁺ cells (±SEM) in Table 13.

Mice challenged with Fel d 1 intranasally when treated with thecombination of anti-Fel d 1 antibodies exhibited a significant decreasein the frequency of eosinophils in the CD45+ cell population as comparedto mice receiving no antibody (67% decrease) or receiving isotypecontrol antibody (46% decrease) as shown in Table 13.

Lung Harvest for Histological Analysis:

After exsanguination, the left lungs were removed and placed into tubescontaining a 5 mL solution of 4% (w/v) paraformaldehyde (BostonBioproducts, # BM-155) in 1× phosphate buffered saline and stored atroom temperature for 3 days. Lung samples were then blotted dry andtransferred to tubes containing 70% ethanol for histological analysis.The samples were sent to Histoserv, Inc (Germantown, Md.) for sectioningand periodic acid Schiff (PAS) staining.

Approximately 35 digital images across the full area of each PAS-stainedlung section were acquired using a Zeiss Axioplan 2 Imaging lightmicroscope with a Zeiss AxioCam MRc camera. A whole lung image was thenconstructed from the smaller images and analyzed using ImageJ softwarewith the aid of a color threshold plugin. The regions of mucusaccumulation in the bronchial lumen were identified and quantitatedthrough a user-chosen color threshold and normalized to the total areaof the lumen that was identified and quantitated by a separate colorthreshold setting. Percentage of the bronchial lumen occupied by mucusaccumulation for each lung was expressed as [(mucus area/lumenarea)×100] and was calculated for each treatment group. Results,expressed as mean percent lung obstruction (±SEM) are shown in Table 14.

Mice treated with the combination of anti-Fel d 1 antibodies exhibited atrend towards reduced mucus accumulation in the lung bronchi(5.21+/−0.81% mucus accumulation) compared to mice receiving controlantibody (10.81+/−1.13% mucus accumulation) in the lung inflammationmodel as shown in Table 14. No differences were observed in bronchiallumen size or overall lung size between the groups of mice.

TABLE 9 Experimental dosing and treatment protocol for groups of Balb/cmice Subcutaneous Intraperitoneal antibody Immunization IntranasalChallenge injection Group (D0) and boost (D7) (D20, D24 & D28) (D17,D21, D25) 1 1 ug Fel d 1 in 1X phosphate No antibody 1 mg Alum bufferedsaline 2 1 ug Fel d 1 in .05 ug/20 uL Fel d 1 No antibody 1 mg Alum 3 1ug Fel d 1 in .05 ug/20 uL Fel d 1 Human IgG4 1 mg Alum isotype control4 l ug Fel d 1 in .05 ug/20 uL Fel d 1 H4H1232N + 1 mg Alum H4H2636P

TABLE 10 Total Circulating IgE levels in Mouse Serum Mean circulatingIgE levels Mouse group (ng/mL) (±SEM) 1. Saline Challenge, no antibodytreatment (n = 19)  2661 (±361)*** 2. Fel d 1 challenge, no antibodytreatment (n = 20) 11711 (±1518) 3. Fel d 1 challenge, human IgG4Isotype control 14080 (±1505) treatment (n = 20) 4. Fel d 1 challenge,anti-Fel d 1 antibody  6683 (±1394)*** treatment (n = 20) Note:Statistical significance compared to isotype control determined byone-way ANOVA with Tukey's multiple comparison post-test is indicated(***p < 0.001). Outliers, defined as greater than 2 standard deviationsfrom the mean, were removed from the study.

TABLE 11 Circulating Fel d 1-specific IgG1 in Mouse Serum Meancirculating Fel d 1 - specific IgG1 levels (Titer × Mouse group 10³)(±SEM) 1. Saline Challenge, no antibody treatment (n = 19) 81.79(±22.07)*** 2. Fel d 1 challenge, no antibody treatment (n = 19) 720.1(±102.8) 3. Fel d 1 challenge, human IgG4 Isotype 526.1 (±144.0) controltreatment (n = 19) 4. Fel d 1 challenge, anti-Fel d 1 antibody 105.3(±31.33)** treatment (n = 19) Note: Statistical significance compared toisotype control determined by one-way ANOVA with Dunn's multiplecomparison post-test is indicated (***p < 0.001, **p < 0.01). Outliers,defined as greater than 2 standard deviations from the mean, wereremoved from the study.

TABLE 12 Antibodies Used for Flow Cytometry Analysis Catalog Concentra-Antibody Fluorochrome Company Number tion CD11c APC BDBiosciences 5502611/100 CD45 PerCP Cy5.5 BDBiosciences 552950 1/800 F4/80 Pacific BlueeBiosciences 48-4801-82 1/200 Siglec-F PE BDBiosciences 552126 1/100Ly6G APC-eFluor780 eBiosciences 47-5931-82 1/200 (Gr-1)

TABLE 13 Frequency of eosinophils in CD45⁺ cells as determined by flowcytometry Mean Frequency of Eosinophils in CD45+ cells Mouse group(±SEM) 1. Saline Challenge, no antibody treatment (n = 19) 1.05(±0.10)*** 2. Fel d 1 challenge, no antibody treatment (n = 20) 6.28(±0.59)** 3. Fel d 1 challenge, human IgG4 Isotype control 3.89 (±0.60)treatment (n = 19) 4. Fel d 1 challenge, anti-Fel d 1 antibody 2.08(±0.23)* treatment (n = 19) Note: Statistical significance compared toisotype control determined by one-way ANOVA with Tukey's multiplecomparison post-hoc test is indicated (***p < 0.001, **p < 0.01, *p <0.05). Outliers, defined as greater than 2 standard deviations from themean, were removed from the analysis.

TABLE 14 Lung Obstruction (mucus area/lumen area, %) Lung ObstructionMouse group (±SEM) 1. Saline Challenge, no antibody treatment (n = 19) 0.48 (±0.10)*** 2. Fel d 1 challenge, no antibody treatment (n = 20)10.31 (±0.75) 3. Fel d 1 challenge, human IgG4 Isotype control 10.18(±1.13) treatment (n = 19) 4. Fel d 1 challenge, anti-Fel d 1 antibody 5.21 (±0.81) treatment (n = 20) Note: Statistical significance comparedto isotype control determined by one-way ANOVA with Dunn's multiplecomparison post-hoc test is indicated (***p < 0.001, **p < 0.01, *p <0.05). Outliers, defined as greater than 2 standard deviations from themean, were removed from the analysis.

Example 8 Hydrogen-Deuterium Exchange Epitope Mapping

In order to determine the epitopes of Fel d1 (a heterodimeric proteincomprised of Fel d1 chain A and FELD1 chain B) recognized by twoanti-Fel d1 antibodies, hydrogen-deuterium (H/D) exchange studies wereperformed for each antibody co-complexed with Fel d1. Prior to the H/Dexchange experiments, CHO cell-expressed recombinant Fel d1 comprised ofamino acids 18-109 of Feld 1 chain B (GenBank accession numberNP_001041619.1) fused in-line with amino acids 19-88 of FELD1 A (GenBankaccession #NP_001041618.1) expressed with a C-terminalmyc-myc-hexahistidine tag and with a D27G mutation (Fel d1 B-A-mmH; SEQID: 396) was deglycosylated at 37° C. for 4 hours under nativeconditions using PNGase F (New England BioLabs, #0704). For this study,two anti-FELD1 antibodies (H4H1232N and H4H2636P) were covalentlyattached to N-hydroxysuccinimide (NHS) agarose beads (GE Lifescience,#17-0906-01) according to the manufacturer's protocol.

To map the Fel d1 B-A-mmH binding epitope recognized by H4H1232N, twosets of H/D exchange experiments were carried out (all binding andexchange reactions carried out at room temperature). The firstexperiment used an ‘on-solution/off-beads’ format (on-exchange insolution followed by off-exchange on beads). For the on-exchange, thedeglycosylated Fel d1 B-A-mmH protein was deuterated for 5 and 10minutes (in two separate sub-experiments) in PBS buffer at pH 7.4prepared with D₂O (PBS-D) and was then bound to the H4H1232N beadsduring a 2-minute incubation in PBS-D. The co-complex of Fel d1B-A-mmH-bound to H4H1232N beads was then washed with PBS buffer at pH7.4 prepared with H₂O (PBS-H) and incubated in PBS-H for half of theon-exchange time (off-exchange), allowing only the epitopes on Fel d1B-A-mmH protected by the binding of the H4H1232N antibody to remaindeuterated. After the off-exchange, the bound Fel d1 B-A-mmH was elutedfrom the beads using an ice-cold 0.1% aqueous trifluoroacetic acid (TFA)solution. The eluted Fel d1 B-A-mmH was then digested with immobilizedpepsin (Thermo Scientific, #20343) for 5 minutes at 4° C. The resultingpeptides were desalted at 4° C. using ZipTip chromatographic pipettetips (Millipore, #ZTC18S096) according to the manufacturer's protocoland then immediately analyzed on an UltrafleXtreme matrix assisted laserdesorption ionization time of flight (MALDI-TOF) mass spectrometer (MS).

The second experiment is referred to as the ‘on-beads/off-beads’(on-exchange on beads followed by off-exchange on beads). For thisexperiment, the deglycosylated Fel d1 B-A-mmH was first bound to theH4H1232N beads, and then incubated for 5 or 10 minutes (in separatesub-experiments) in PBS-D to allow on-exchange. The following steps(off-exchange, pepsin digestion, and MS analysis) were carried out asdescribed for the ‘on-solution/off-beads’ procedure above. The centroidvalues or average mass-to-charge ratios (m/z) of all the detectedpeptides were calculated and compared between the on-solution/off-beadsand on-beads/off-beads experiments. Peptides exhibiting increased massafter the on-solution/off-beads procedure compared to theon-beads/off-beads procedure include amino acids within the Fel d1protein protected from exchange as a result of antibody binding andtherefore reveal binding epitope regions.

The H/D exchange experiment for Fel d1 B-A-mmH binding to the anti-Feld1 antibody H4H2636P was performed using the same procedure describedabove for H4H1232N, but with H4H2636P beads replacing the H4H1232Nbeads.

A comparison of the centroid m/z values for all the peptides detected inthe H/D exchange experiment of Fel d1 B-A-mmH with H4H1232N are shown inTable 15. These peptides were identified by liquid chromatography-matrixassisted laser desorption ionization (LC-MALDI) MS. Most peptic peptidesgave similar centroid values (differences <0.3 m/z units) for both theon-solution/off-beads and on-beads/off-beads protocols, for each of twodifferent on-exchange and off-exchange times. However, three peptideswith amino acids spanning from 85-103, 85-104, and 113-127 of Feld1B-A-mmH (SEQ ID NO: 396) had differences in m/z centroid values >0.3in both the 5 minute and 10 minute experiments. The differences betweenthese centroid values from the on-solution/off-beads andon-beads/off-beads protocol are highlighted in bold in Table 15. Sinceanother peptide, amino acids 117-127 of SEQ ID NO: 396, did not showdeuteron retention after off-exchange, the region of protection fromexchange in the 113-127 peptide can be reduced to residues 113-116 ofSEQ ID NO: 396. The two regions, residues 85-104 (SEQ ID NO: 403) and113-116 (SEQ ID NO: 426), are protected from full off-exchange as aresult of H4H1232N binding to Fel d1 B-A-mmH after on-exchange.Therefore, these two segments are defined by the H/D exchange method asa discontinuous epitope for antibody H4H1232N binding to the Fel d1B-A-mmH protein.

Comparisons of the centroid m/z values for the peptides detected in theH/D exchange experiment of Fel d1 B-A-mmH complexed with H4H2636P areshown in Table 16. Only one peptide, amino acids 15-24 of FELD1 B-A-mmH,exhibited an increase in the centroid m/z values >0.3 m/z for theon-solution/off-beads condition compared to the on-beads/off-beadscondition, indicating that this segment was protected from fulloff-exchange by the binding of H4H2636P. The centroid value differencesgreater than 0.3 m/z are highlighted in bold in Table 16. Therefore,amino acids within this 15-24 region (SEQ ID NO: 412) based on the H/Dexchange method include an epitope for antibody H4H2636P binding to theFel d1 B-A-mmH protein.

TABLE 15 The Effect on H/D Exchange of H4H1232N Binding Fel d1B-A-mmH asMeasured by Centroid m/z Values of Peptic Peptides Experiment IExperiment II 5 min on-/2.5 min off- 10 min on-/5 min off- exchangeexchange Residues on- on- of on- on- solution/ beads/ FELDB- solution/beads/ off off- A-MMH off beads off-beads D beads beads D Peptide   1-111318.37 1318.29 0.09 1318.27 1318.27 -0.01 VKMAETCPIFY (SEQ ID NO: 398) 55-61 759.89 759.83 0.06 759.87 759.86 0.01 ISRVLDG (SEQ ID NO: 399) 55-62 873.04 873.02 0.02 873.02 873 0.02 ISRVLDGL (SEQ ID NO: 400) 55-64 1103.37 1103.39 -0.03 1103.36 1103.36 -0.01 ISRVLDGLVM(SEQ ID NO: 401)  85-103 2084.38 2083.75 0.63 2084.28 2083.83 0.45LKLNTLGREICP AVKRGVD (SEQ ID NO: 402)  85-104 2197.63 2196.99 0.642197.73 2197.21 0.52 LKLNTLGREICP AVKRGVDL (SEQ ID NO: 403) 113-1271721.91 1721.33 0.58 1722.22 1721.53 0.69 YVEQVAQYKAL PVVL (SEQ IDNO: 404) 117-127 1201.47 1201.48 -0.01 1201.56 1201.46 0.1 VAQYKALPVVL(SEQ ID NO: 405) 128-141 1606.43 1606.26 0.16 1606.55 1606.29 0.26ENARILKNCVDA KM (SEQ ID NO: 406) 153-170 1920.33 1920.25 0.09 1920.371920.38 -0.01 LDKIYTSPLCGP GGEQKL (SEQ ID NO: 407) 183-196 1672.541672.59 -0.04 1672.54 1672.51 0.03 ISEEDLSGHHHH HH (SEQ ID NO: 408)183-199 1903.91 1903.95 -0.03 1903.95 1903.92 0.03 ISEEDLSGHHHHHHSSG (SEQ ID NO: 409) 186-199 1574.44 1574.44 0.01 1574.44 1574.47-0.03 EDLSGHHHHHH SSG (SEQ ID NO: 410)

TABLE 16The Effect on H/D Exchange of H4H2636P Binding to Fel d1B-A-mmH as Measuredby Centroid m/z Values of Peptic Peptides Experiment I Experiment II5 min on-/2.5 min off- 10 min on-/5 min off- exchange exchange Residueson- on- of on- on- solution/ beads/ FELDB- solution/ beads/ off off-A-MMH off beads off-beads D beads beads D Peptide   1-11 1318.47 1318.48-0.01 1318.5 1318.47 0.03 VKMAETCPIFY (SEQ ID NO: 411)  15-24 1049.531048.6 0.94 1049.51 1048.71 0.79 FAVANGNELL (SEQ ID NO: 412)  55-61759.85 759.89 -0.04 759.87 759.88 -0.01 ISRVLDG (SEQ ID NO: 413)  55-62873.06 873.01 0.05 873.06 873.04 0.02 ISRVLDGL (SEQ ID NO: 414)  55-641103.39 1103.36 0.03 1103.42 1103.43 -0.01 ISRVLDGLVM (SEQ ID NO: 415) 85-103 2083.63 2083.63 0 2083.67 2083.62 0.04 LKLNTLGREICP AVKRGVD (SEQID NO: 416)  85-104 2196.67 2196.74 -0.07 2196.8 2196.78 0.02LKLNTLGREICP AVKRGVDL (SEQ ID NO: 417) 113-127 1721.28 1721.27 0.011721.19 1721.21 -0.02 YVEQVAQYKAL PVVL (SEQ ID: 418) 117-127 1201.551201.53 0.02 1201.55 1201.59 -0.04 VAQYKALPVVL (SEQ ID NO: 419) 120-127903.18 903.12 0.06 903.14 903.14 -0.01 YKALPVVL (SEQ ID NO: 420) 128-1411606.41 1606.34 0.08 1606.57 1606.46 0.11 ENARILKNCVDA KM (SEQ ID NO:421) 153-170 1920.29 1920.23 0.06 1920.24 1920.36 -0.13 LDKIYTSPLCGPGGEQKL (SEQ ID NO: 422) 183-196 1672.56 1672.59 -0.02 1672.58 1672.540.04 ISEEDLSGHHHH HH (SEQ ID NO: 423) 183-199 1903.9 1903.89 0.011903.94 1903.93 0.02 ISEEDLSGHHHH HHSSG (SEQ ID NO: 424) 186-199 1574.41574.37 0.03 1574.41 1574.37 0.04 EDLSGHHHHHH SSG (SEQ ID NO: 425)

Example 9 Generation of Bi-Specific Antibodies

Description of the Fel d 1 Bispecific Antibodies Produced

Bi-specific antibodies comprising heavy and light chain binding domainsfrom pairs of certain of the anti-Fel d1 antibodies described in thepresent invention were constructed using standard methodologies. Theanti-Fel d1 antibodies used to construct the bi-specific antibodies ofthis example were obtained by immunizing a Veloclmmune® mouse with aprimary immunogen, such as full length natural Fel d1, which may bepurchased commercially (e.g., from Indoor Biotechnologies, # LTN-FD1-1),or isolated from cat hair or dander by multi-step column chromatography(See, for example, Chapman M D, et al. (1988), J. Immunol. 140:812-818),or which may be produced recombinantly (See GenBank accession numbersP30438, or NP_001041618.1 for the full length amino acid sequence ofchain 1 of Fel d1 (also referred to as chain A or FELD1 A; also see SEQID NO: 392) and GenBank accession number P30440, or NP_001041619.1 forthe full length amino acid sequence of chain 2 of Fel d1 (also referredto as chain B or FELD B; also see SEQ ID NO: 393), or fragments ofeither chain 1 or chain 2, or fragments from both chain 1 and chain 2 ofthe Fel d1 protein, followed by immunization with a secondary immunogen,or with an immunogenically active fragment of the natural protein. Inone embodiment, the immunogen used is exemplified in SEQ ID NO: 394 (inline fusion of Fel d1 Chain-2-Chain1-mFc) or SEQ ID NO: 395 (fusion ofFel d1 Chain 1 using a linker and Chain 2-mFc).

The bi-specific antibodies produced in accordance with the presentExample comprise two antigen-binding domains (i.e. “binding arms 1 and2”).

One of the bi-specific antibodies, designated H4H3467D comprises acommon kappa light chain on both Fab arms, derived from the antibodyH4H2864P (SEQ ID NO: 378). One Fab arm of H4H3467D utilizes the heavychain variable region (V_(H)) from the antibody H4H2864P (SEQ ID NO:370), while the other Fab arm utilizes the V_(H) region from H4H1232N(SEQ ID NO: 18).

A second bi-specific antibody of the invention, designated H4H8751 D,comprises a common kappa light chain on both Fab arms, derived from theantibody H4H2636P (SEQ ID NO: 314). One Fab arm of H4H8751 D utilizesthe V_(H) region from H4H2636P (SEQ ID NO: 306), while the other Fab armutilizes the V_(H) region from H4H1232N (SEQ ID NO: 18).

Table 17 below provides the component parts of the antigen-bindingdomains of the two bi-specific antibodies made in accordance withExample 9. The amino acid sequence identifiers for the various heavychain and light chain variable regions that were derived from theparental antibodies (used to prepare the bi-specific antibodies) arealso provided in Table 17.

TABLE 17 Component parts of the two arms of the bi-specific antibodiesproduced Parental Antibody Identifier from which Bi-specific SequenceDerived Arm 1 Antigen Arm 2 Antigen Bispecific Binding Domain BindingDomain Identifier HCVR LCVR HCVR LCVR H4H3467D H4H2864P H4H2864PH4H1232N H4H2864P SEQ ID SEQ ID SEQ ID SEQ ID NO: 370 NO: 378 NO: 18 NO:378 H4H8751D H4H2636P H4H2636P H4H1232N H4H2636D SEQ ID SEQ ID SEQ IDSEQ ID NO: 306 NO: 314 NO: 18 NO: 314

Tables 18A and 18B below set forth the amino acid sequence identifiersfor the various heavy chain variable regions (Table 18A) and the lightchain variable regions (Table 18B) and their correspondingcomplementarity determining region sequences (CDRs) for the twobi-specific antibodies described herein.

TABLE 18A HCVR and HCDR Sequence Identifiers for bi-specific antibodiesproduced Parent Ab from which Bi-specific Ab sequences SEQ ID NOsIdentifier derived HCVR HCDR1 HCDR2 HCDR3 H4H3467D H4H2864P 370 372 374376 (Arm 1) H4H1232N 18 20 22 24 (Arm 2) H4H8751D H4H2636P 306 308 310312 (Arm 1) H4H1232N 18 20 22 24 (Arm 2)

TABLE 18B LCVR and LCDR Sequence Identifiers for bi-specific antibodiesproduced Parent Ab from which Bi-specific Ab sequences SEQ ID NOsIdentifier derived LCVR LCDR1 LCDR2 LCDR3 H4H3467D H4H2864P 378 380 382384 (Arm 1) H4H2864P 378 380 382 384 (Arm 2) H4H8751D H4H2636P 314 316318 320 (Arm 1) H4H2636P 314 316 318 320 (Arm 2)Biacore Analysis of Bi-Specific Antibodies to Determine Association andDissociation Values

Binding association and dissociation rate constants (k_(a) and k_(d),respectively), equilibrium dissociation constants and dissociationhalf-lives (K_(D) and t_(1/2), respectively) for natural Fel d 1(subsequently referred to as nFel d 1) binding to purified anti-Fel d 1monospecific and bispecific antibodies were determined using a real-timesurface plasmon resonance biosensor assay on a Biacore 2000 instrument.On a CM5 chip, using the EDC-NHS chemistry, the Biacore sensor surfacewas derivatized with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture anti-Fel d 1 monospecific and bispecificantibodies. All the Biacore binding studies were performed at 25° C. inHBSP+ running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3 mM CaCl₂, 3 mMMgCl₂, 0.05% v/v Surfactant P20). Different concentrations of nFel d 1(Indoor Biotech, # NA-FD1-2) (ranging from 600 nM to 2.34 nM, 6-folddilutions) prepared in HBSP+ running buffer were injected over theanti-Fel d 1 antibody captured surface at a flow rate of 50 μL/min.Association of nFel d 1 to the captured monoclonal antibodies wasmonitored for 4 minutes and the dissociation of nFel d 1 in HBSP+running buffer was monitored for 7 minutes. Kinetic association (k_(a))and dissociation (k_(d)) rate constants were determined by fitting thereal-time sensorgrams to a 1:1 binding model with mass transportlimitation using Scrubber 2.0c curve fitting software. Bindingdissociation equilibrium constants (K_(D)) and dissociative half-lives(t₁₂) were then calculated from the kinetic rate constants as: K_(D)(M)=k_(d)/k_(a) and t_(1/2) (min)=[In2/(60*k_(d))].

Binding kinetics of nFel d1 binding to different anti-Fel d 1mono-specific and bi-specific antibodies at 25° C. are shown in Table19. The three monospecific anti-Fel d 1 antibodies bound to nFel d1 withK_(D) values ranging from 155 pM to 1.6 nM. The two bi-specific anti-Feld1 antibodies, H4H3467D and H4H8751D, bound to nFel d1 with K_(D) valuesof 250 pM and 347 pM respectively.

TABLE 19 Binding Kinetics of anti-Fel d1 mono-specific and bi-specificantibodies binding to nFel d 1 at 25° C. Amount of 600 nM mAb CapturednFel d 1 k_(a) k_(d) K_(D) t½ AbPID (RU) Bound (RU) (1/Ms) (1/s) (M)(min) H4H2864N 277 48 9.16E+05 9.47E−04 1.03E−09 12 H4H2636N 280 543.14E+05 5.02E−04 1.60E−09 23 H4H1232N 259 42 3.05E+06 4.72E−04 1.55E−1024 H4H3467D 278 35 2.85E+06 7.12E−04 2.50E−10 16 H4H8751D 300 301.79E+06 6.20E−04 3.47E−10 19

To determine the in vivo efficacy of the anti-Fel d 1 bi-specificscompared with their mono-specific parental antibodies, these antibodiesalong with an isotype control antibody were tested in the PCA in vivomodel using natural Fel d 1 for both sensitization and challenging,which was previously described (see Example 6). Antibodies in this studywere administered at a concentration of 1 mg/kg total antibody (0.5mg/kg of each antibody was used when two antibodies were administeredsimultaneously) using 8 mice per experimental group. The data for eachexperimental group expressed as percent reduction in dyeextravasation±SD are shown in Table 20.

The mono-specific antibodies H4H1232N and H4H2864P caused a 67 (±26)%and an 81 (±26)% reduction in dye extravasation, respectively. Thecombination of the mono-specific antibodies, H4H1232N and H4H2864P,caused a 98 (±3.5)% reduction in dye extravasation, while thebi-specific, H4H3467D, composed of the mono-specific antibodies,H4H1232N and H4H2864P, caused a 93 (±11)% reduction in dyeextravasation.

The mono-specific antibodies H4H1232N and H4H2636P caused a 64 (±33)%and an 8.7 (±79)% reduction in dye extravasation, respectively, inanother experiment. The combination of the mono-specific antibodies,H4H1232N and H4H2636P, caused a 90 (±15)% reduction in dyeextravasation, while the bi-specific, H4H8751 D, composed of themono-specific antibodies, H4H1232N and H4H2636P, caused a 77 (±20)%reduction in dye extravasation.

TABLE 20 Effect of anti-Fel d 1 bispecific antibodies and their parentalmono-specific antibodies in the passive cutaneous anaphylaxis (PCA) invivo model % Reduction in Dye Antibody Extravasation ± SD H4H1232N 67 ±26**** H4H2864P 81 ± 26**** H4H3467D 93 ± 11**** H4H1232N + H4H2864P  98± 3.5**** H4H1232N^(a) 64 ± 33***  H4H2636P^(a) 8.7 ± 79    H4H8751D^(a)77 ± 20**** H4H1232N + H4H2636P^(a) 90 ± 15**** ^(a)Experimentsperformed on a separate day Statistical significance compared to isotypecontrol determined by two-way ANOVA with Bonferroni's multiplecomparison post-test is indicated (***= p < 0.001 and ****= p < 0.00001)

What is claimed is:
 1. A nucleic acid molecule encoding a humanmonoclonal antibody, or a fragment thereof that binds specifically toFel d1, wherein the antibody comprises the three heavy chaincomplementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3)contained within a heavy chain variable region (HCVR) amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274,290, 306, 322, 338, 354 and 370; and the three light chain CDRs (LCDR1,LCDR2 and LCDR3) contained within the light chain variable region (LCVR)amino acid sequence selected from the group consisting of SEQ ID NOs:10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234,250, 266, 282, 298, 314, 330, 346, 362 and
 378. 2. The nucleic acidmolecule of claim 1, wherein the nucleic acid encodes an antibody orfragment thereof comprising a HCVR having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82,98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306,322, 338, 354 and
 370. 3. The nucleic acid molecule of claim 1, whereinnucleic acid encodes the antibody or fragment thereof comprising a LCVRhaving an amino acid sequence selected from the group consisting of SEQID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218,234, 250, 266, 282, 298, 314, 330, 346, 362 and
 378. 4. The nucleic acidmolecule of claim 1, wherein nucleic acid encodes the antibody orfragment thereof comprising a HCVR/LCVR amino acid sequence pair of SEQID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250,258/266, 274/282, 290/298, 306/314, 322/330, 338/346, 354/362 and370/378.
 5. An expression vector comprising the nucleic acid moleculeencoding a human monoclonal antibody, or a fragment thereof that bindsspecifically to Fel d1, according to claim
 1. 6. A method of producing aFel d1 antibody or antigen-binding fragment thereof comprising the stepsof introducing the expression vector of claim 5 into an isolated hostcell, growing the cell under conditions permitting production of theantibody or antibody fragment, and recovering the antibody or antibodyfragment so produced.
 7. The method of claim 6, wherein the host cell isa CHO cell.
 8. An isolated host cell containing the expression vector ofclaim 5.